Replication Is Connected to the Cell Cycle

© Jones & Bartlett Learning,Part LLC © JonesII & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION DNA Replication © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION and NOTRecombination FOR SALE OR DISTRIBUTION

© Jones & BartlettChap Learning,ter 9 ReplicationLLC Is Connected© Jonesto the Cell& Bartlett Cycle Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Chapter 10 The Replicon: Initiation of Replication Chapter 11 DNA Replication

© Jones & Bartlett Learning,Cha LLCpter 12 Extrachromosomal© Jones &Replicons Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONChapter 13 HomologousNOT and FOR Site-Specific SALE OR DISTRIBUTION Recombination Chapter 14 Repair Systems © Jones & Bartlett Learning, LLC Chapter 15© Jones Transposable & Bartlett Elemen Learning,ts and LLC Retroviruses NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Chapter 16 Somatic Recombination and Hypermutation in the Immune System

9781284104646_CH09_227_244.indd 227 01/02/17 5:09 PM © Jones & Bartlett Learning LLC, an Ascend Learning Company. NOT FOR SALE OR DISTRIBUTION. Chapter 9 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Replication Is Connected © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC to theNOT Cell FOR SALE ORCycle DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Edited by Barbara Funnell

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC ChaNOTpte rFOR Outli SALEne OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 9.1 Introduction 9.7 Partition Involves Separation of the 9.2 Bacterial Replication Is Connected to the Chromosomes Cell Cycle 9.8 Chromosomal Segregation Might Require Site-Specific Recombination © Jones &9.3 Bartlett The Shape Learning, and Spatial LLC Organization © Jones & Bartlett Learning, LLC of a Bacterium Are Important During 9.9 The Eukaryotic Growth Factor Signal NOT FOR SALEChromosome OR DISTRIBUTION Segregation and Cell Division NOT FORTransduction SALE OR DISTRIBUTION Pathway Promotes Entry 9.4 Mutations in Division or Segregation Affect to S Phase Cell Shape 9.10 Checkpoint Control for Entry into S Phase: 9.5 FtsZ Is Necessary for Septum Formation p53, a Guardian of the Checkpoint © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 9.6 min and noc/slm Genes Regulate the 9.11 Checkpoint Control for Entry into S Phase: Location of theNOT Septum FOR SALE OR DISTRIBUTIONRb, a Guardian of theNOT Checkpoint FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC • Replication© Jones requires & Bartlett coordination Learning, of these replicons LLC 9.1 Introduction to reproduce DNA during a discrete period of the NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION A major difference between prokaryotes and eukaryotes is the cell cycle. way in which replication is controlled and linked to the cell cycle. • The decision about whether to replicate is determined In eukaryotes, the following are true: by a complex pathway that regulates the cell cycle. • Duplicated chromosomes are segregated to daugh- • Chromosomes reside in the nucleus. ter cells during mitosis by means of a special © Jones & Bartlett• Each Learning,chromosome consistsLLC of many units of repli-© Jones & Bartlett Learning, LLC apparatus. NOT FOR SALEcation OR calledDISTRIBUTION replicons. NOT FOR SALE OR DISTRIBUTION

228 Top texture: © Laguna Design / Science Source; Chapter Opener: © Laguna Design/Science Source.

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Mother A unit cell has a circular chromosome © Jones & Bartlett Learning, LLC S © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONG1 NOT FORReplication SALE ORinitiates DISTRIBUTION when cell passes critical size Divide Mitosis G2 M Replication generates Daughter Daughter catenated daughter © Jones & BartlettStart Learning, LLC chromosomes © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Daughter chromosomes Grow Interphase = G1 + S + G2 are separated

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION SeptumNOT divides FOR cell SALE OR DISTRIBUTION Figure 9.1 A growing cell alternates between cell division of a mother cell into two daughter cells and growth back to the original size. Daughter cells separate © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Figure 9.2 Replication initiates at the bacterial origin when a NOT FOR SALEIn eukaryotic OR DISTRIBUTION cells, replication of DNA is confined to theNOT cellFOR passes SALE a critical OR threshold DISTRIBUTION of size. Completion of replication second part of the cell cycle called S phase, which follows G1 produces daughter chromosomes that might be linked by phase (see Figure 9.1). The eukaryotic cell cycle is composed recombination or that might be catenated. They are separated and moved to opposite sides of the septum before the bacterium of alternating rounds of growth followed by DNA replication is divided into two. and then cell division. After the cell divides into two daughter cells, each has the option© Jones to continue & Bartlett dividing or Learning, stop and LLC © Jones & Bartlett Learning, LLC enter G0. If the decisionNOT is to continue FOR SALEto divide, OR the cellDISTRIBUTION must NOT FOR SALE OR DISTRIBUTION grow back to the size of the original parent cell before divi- would trigger initiation. This is consistent with the fact that sion can occur again. protein synthesis is needed for the initiation event. Another The G1 phase of the cell cycle is concerned primarily possibility is that an inhibitor protein might be synthesized with growth (although G1 is an abbreviation for first gap or activated at a fixed point and then diluted below an effec- because© Jonesthe early &cytologists Bartlett could Learning, not see any LLC activity). In tive level by© theJones increase & Bartlettin cell volume. Learning, Current LLCmodels G1 everythingNOT FOR except SALE DNA beginsOR DISTRIBUTION to be doubled: RNA, pro- suggest thatNOT variations FOR of SALE both possibilities OR DISTRIBUTION operate to turn tein, lipids, and carbohydrates. The progression from G1 into initiation on and then off precisely in each cell cycle. Syn- S is very tightly regulated and is controlled by a checkpoint. thesis of active DnaA protein, the bacterial initiator protein, For a cell to be allowed to progress into S phase, there must reaches a threshold that turns on initiation, and the activity be a certain minimum amount of growth that is biochemi- of inhibitors turns subsequent initiations off for the rest of © Jones &cally Bartlett monitored. Learning, In addition, LLC there must not be any damage© Jonesthe cell & cycle. Bartlett This is Learning, described in the LLC The Replicon: Initiation NOT FORto SALE the DNA. OR Damaged DISTRIBUTION DNA or too little growth prevents theNOT ofFOR Replication SALE chapter OR .DISTRIBUTION cell from progressing into S phase. When S phase is com- Bacterial chromosomes are specifically compacted and plete, G2 phase commences; there is no control point and no arranged inside the cell, and this organization is important sharp demarcation. for proper segregation, or partition, of daughter chromo- The start of S phase is signaled by the activation of the somes at cell division. Some of the events in partitioning the first replicon—usually ©in Joneseuchromatin—in & Bartlett areas Learning,of active LLCdaughter chromosomes are consequences© Jones & of Bartlett the circularity Learning, LLC genes. Over the next fewNOT hours, FOR initiation SALE events OR occurDISTRIBUTION at of the bacterial chromosome.NOT Circular FOR chromosomes SALE OR are DISTRIBUTION other replicons in an ordered manner. said to be catenated when one passes through another, con- However, replication in bacteria, as shown in Figure 9.2, necting them. Catenation is a consequence of incomplete is triggered at a single origin when the cell mass increases removal of topological links during DNA replication, and past a threshold level, and the segregation of the daughter topoisomerases are required to remove these links and sep- chromosomes© Jones is &accomplished Bartlett Learning,by ensuring thatLLC they find arate the chromosomes.© Jones & An Bartlett alternative Learning, type of structure LLC is themselvesNOT onFOR opposite SALE sides OR of DISTRIBUTIONthe septum that grows to formed whenNOT a recombination FOR SALE event OR occurs: DISTRIBUTION A single recom- divide the bacterium into two. bination between two monomers converts them into a single How does the cell know when to initiate the replication dimer. This is resolved by a specialized recombination sys- cycle? The initiation event occurs once in each cell cycle tem that recreates the independent monomers. and at the same time in every cell cycle. How is this timing The key goals in the chapters that follow are to define © Jones &set? Bartlett An initiator Learning, protein could LLC be synthesized continuously© Jonesthe DNA & Bartlett sequences thatLearning, function in LLC replication and to deter- NOT FORthroughout SALE OR the cellDISTRIBUTION cycle; accumulation of a critical amountNOT mineFOR how SALE they ORare recognizedDISTRIBUTION by appropriate proteins of

9.1 Introduction 229

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the replication apparatus. In subsequent chapters, we exam- Origin Terminus ine the unit of replication and how that unit is regulated to © Jones start& Bartlett replication; Learning, the biochemistry LLC and mechanism of DNA© Jones & Bartlett Learning, LLC NOT FORsynthesis; SALE andOR autonomouslyDISTRIBUTION replicating units in bacteria,NOT FOR SALE OR DISTRIBUTIONDivision mitochondria, and chloroplasts. 35/0 30 5 9.2 Bacterial Replication Is Connected t©o Jones the Cell& Bartlett Cycle Learning, LLC 25 ©Initiation Jones10 & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 20 15 Key Concepts Termination • The doubling time of Escherichia coli can vary over a range of© up Jones to 10 times, & dependingBartlett on Learning, growth conditions. LLC Figure 9.3 ©The Jones fixed interval & Bartlett of 60 minutes Learning, between initiation LLC of • It NOTrequires FOR 40 minutes SALE to replicate OR DISTRIBUTION the bacterial replication andNOT cell divisionFOR producesSALE ORmultiforked DISTRIBUTION chromosomes chromosome (at normal temperature). in rapidly growing cells. Note that only the replication forks • Completion of a replication cycle triggers a bacterial moving in one direction are shown; the chromosome actually is division 20 minutes later. replicated symmetrically by two sets of forks moving in opposite directions on circular chromosomes. • If the doubling time is approximately 60 minutes, a © Jones & Bartlettreplication cycleLearning, is initiated LLC before the division resulting © Jones & Bartlett Learning, LLC from the previous replication cycle. NOT FOR SALE• Fast rates OR of growthDISTRIBUTION therefore produce multiforked NOT FORA cycleSALE of chromosome OR DISTRIBUTION replication must be initiated at chromosomes. a fixed time of C + D = 60 minutes before cell division. For bacteria dividing more frequently than every 60 minutes, a cycle of replication must be initiated before the end of the Bacteria have two links between replication and cell growth: preceding division cycle. You might say that a cell is born • The frequency© of initiationJones &of cyclesBartlett of replication Learning, is LLC“already pregnant” with the next© Jones generation. & Bartlett Learning, LLC adjusted to fit NOTthe rate FOR at which SALE the cell OR is growing. DISTRIBUTIONConsider the example of cellsNOT dividing FOR every SALE 35 minutes. OR DISTRIBUTION • The completion of a replication cycle is connected The cycle of replication connected with a division must have with division of the cell. been initiated 25 minutes before the preceding division. This The rate of bacterial growth is assessed by the doubling situation is illustrated in Figure 9.3, which shows the chro- time, the period required for the number of cells to double. mosomal complement of a bacterial cell at 5-minute inter- The shorter© Jones the doubling& Bartlett time, Learning, the faster the LLC bacteria are vals throughout© Jones the cycle. & Bartlett Learning, LLC growing.NOT E. coliFOR growth SALE rates OR can rangeDISTRIBUTION from doubling times At divisionNOT (35/0 FOR minutes), SALE the OR cell DISTRIBUTION receives a partially as fast as 18 minutes to slower than 180 minutes. The bacte- replicated chromosome. The replication fork continues to rial chromosome is a single replicon; thus, the frequency of advance. At 10 minutes, when this “old” replication fork has replication cycles is controlled by the number of initiation not yet reached the terminus, initiation occurs at both origins events at the single origin. Researchers can define the repli- on the partially replicated chromosome. The start of these © Jones &cation Bartlett cycle in Learning, terms of two constants:LLC © Jones“new” & replication Bartlett forks Learning, creates a multiforked LLC chromosome. NOT FOR SALE• C OR is the DISTRIBUTION fixed time of approximately 40 minutesNOT FORAt SALE15 minutes—that OR DISTRIBUTION is, at 20 minutes before the next required to replicate the entire E. coli chromosome. division—the old replication fork reaches the terminus. Its Its duration corresponds to a rate of replication fork arrival allows the two daughter chromosomes to separate; movement of approximately 50,000 bp/minute. each of them has already been partially replicated by the new (The rate of DNA synthesis is more or less invariant replication forks (which now are the only replication forks). at a constant temperature;© Jones &it proceedsBartlett at Learning,the same LLCThese forks continue to advance.© Jones & Bartlett Learning, LLC speed unless andNOT until FOR the SALEsupply ofOR precursors DISTRIBUTION At the point of division,NOT the twoFOR partially SALE replicated OR DISTRIBUTION becomes limiting.) chromosomes segregate. This recreates the point at which we • D is the fixed time of approximately 20 minutes that started. The single replication fork becomes “old,” it termi- elapses between the completion of a round of replica- nates at 15 minutes, and 20 minutes later, there is a division. 25 tion and the cell division with which it is connected. We see that the initiation event occurs 1 /35 cell cycles before © JonesThis period & Bartlett might represent Learning, the time LLC required to the division© event Jones with which& Bartlett it is associated. Learning, LLC NOTassemble FOR the SALE components OR DISTRIBUTION needed for division. The generalNOT principle FOR ofSALE the link OR between DISTRIBUTION initiation and the The constants C and D can be viewed as representing cell cycle is that as cells grow more rapidly (the cycle is shorter), the maximum speed with which the bacterium is capable of the initiation event occurs at an increasing number of cycles completing these processes. They apply for all growth rates before the related division. There are correspondingly more between doubling times of 18 and 60 minutes, but both con- chromosomes in the individual bacterium. This relationship © Jones &stant Bartlett phases becomeLearning, longer LLC when the cell cycle occupies© Jonescan be & viewed Bartlett as the Learning,cell’s response toLLC its inability to reduce the NOT FORmore SALE than OR60 minutes. DISTRIBUTION NOT periodsFOR SALEof C and DOR to keepDISTRIBUTION pace with the shorter cycle.

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chromosome is compacted into a dense protein–DNA struc- 9.3 The Shape and Spatial ture called the nucleoid, which takes up most of the space © Jones O& rganizationBartlett Learning, of LLC a Bacterium © Jonesinside & the Bartlett cell. It is not Learning, a disorganized LLC mass of DNA; instead, NOT FOR SALE OR DISTRIBUTION NOT specificFOR SALEDNA regions OR DISTRIBUTIONare localized to specific regions in the Are Important During cell, and this positioning depends on the cell cycle and on Chromosome Segregation the bacterial species. The movement apart of newly replicated bacterial chromosomes—that is, the segregation of the and Cell Division chromosomes—occurs concurrently with DNA replication. © Jones & Bartlett Learning, LLCFigure 9.4 summarizes the arrangement© Jones &in E.Bartlett coli. In new Learning,- LLC Key ConceptsNOT FOR SALE OR DISTRIBUTIONborn cells, the origin and terminusNOT FORregions SALE of the chromo OR DISTRIBUTION- some are at mid-cell. Following initiation, the new origins • Bacterial chromosomes are specifically arranged and move toward the poles, or the one-quarter and three-quarters positioned inside cells. positions, and the terminus remains at mid-cell. Following • A rigid peptidoglycan cell wall surrounds the cell and gives cell division, the origins and termini reorient to mid-cell. it ©its shape.Jones & Bartlett Learning, LLC The shape© Jones of a bacterial & Bartlett cell is establishedLearning, by LLCa rigid • TheNOT rod shape FOR of SALEE. coli is dependentOR DISTRIBUTION on MreB, PBP2, layer of peptidoglycanNOT FOR in SALEthe cell wall,OR whichDISTRIBUTION surrounds the and RodA. inner membrane. The peptidoglycan is made by polymeriza- • Septum formation is initiated mid-cell, 50% of the distance tion of tri- or pentapeptide-disaccharide units in a reaction from the septum to each end of the bacterium. involving connections between both types of subunit (trans- peptidation and transglycosylation). Three proteins that are © Jones &The Bartlett shape of bacterialLearning, cells LLCvaries among different species,© Jonesrequired & Bartlettto maintain Learning, the rodlike shape LLC of bacteria are MreB, NOT FORbut SALE many, ORincluding DISTRIBUTION E. coli cells, are shaped like cylindri-NOT PBP2,FOR and SALE RodA. OR Mutations DISTRIBUTION in any one of their genes and/or cal rods that end in two curved poles. Bacterial cells have depletion of one of these proteins cause the bacterium to lose an internal cytoskeleton that is similar to what is found its extended shape and become round. in eukaryotes. There are low homology homologs of The structure of MreB protein resembles that of actin, tubulin, and intermediate filaments. The bacterial the eukaryotic protein actin, which polymerizes to form © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

ori Origin and terminus at mid-cell in © Jones & Bartlett Learning, LLCter © Jonesnewborn & Bartlett cells Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Origins and termini reorient and cell divides © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORFigu SALEre 9.4 ORAttachment DISTRIBUTION of bacterial DNA to the membrane couldNOT provide FOR a mechanism SALE for OR segregation. DISTRIBUTION

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cytoskeletal filaments in eukaryotic cells. In bacteria, MreB polymerizes and appears to move dynamically around the cir- © Jones cumference& Bartlett of theLearning, cell attached LLC to the peptidoglycan synthesis© Jones & Bartlett Learning, LLC NOT FORmachinery, SALE OR including DISTRIBUTION PBP2. These interactions are necessaryNOT FOR SALE OR DISTRIBUTION for the lateral integrity of the cell walls, because the lack of MreB results in round, rather than rod-shaped, cells. RodA is a member of the SEDS (shape, elongation, division, and sporulation) family present in all bacteria that have a peptidoglycan cell wall. Each SEDS protein© Jones functions & together Bartlett with Learning, a specific LLC © Jones & Bartlett Learning, LLC transpeptidase, which catalyzesNOT FORthe formation SALE of ORthe crosslinks DISTRIBUTION NOT FOR SALE OR DISTRIBUTION in the peptidoglycan. PBP2 (penicillin-binding protein 2) is the transpeptidase that interacts with RodA. This demonstrates the important principle that shape and rigidity can be determined by the simple extension of a polymeric structure. The© Jonesend of the & cellBartlett cycle in Learning, a bacterium LLCis defined by Figure 9.5 ©Top Jones panel: Wild-type & Bartlett cells. Bottom Learning, panel: Failure LLC the divisionNOT ofFOR a mother SALE cell intoOR two DISTRIBUTION daughter cells. Bacteria of cell divisionNOT under FOR nonpermissive SALE temperatures OR DISTRIBUTION generates divide in the center of the cell by the formation of a septum, multinucleated filaments. a structure that forms in the center of the cell as an invagi- Photos courtesy of Sota Hiraga, Kyoto University. nation from the surrounding envelope. The septum forms an impenetrable barrier between the two parts of the cell © Jones &and Bartlett provides theLearning, site at which LLC the two daughter cells even-© Jones & Bartlett Learning, LLC NOT FORtually SALE separate OR entirely.DISTRIBUTION The septum then becomes the newNOT FOR SALE OR DISTRIBUTION pole of each daughter cell. The septum consists of the same components as the cell envelope. The septum initially forms as a double layer of peptidoglycan, and the protein EnvA is required to split the covalent links between the layers so that the daughter cells ©can Jones separate. & Two Bartlett related Learning,questions LLC © Jones & Bartlett Learning, LLC address the role of the septumNOT inFOR division: SALE “What OR determines DISTRIBUTION NOT FOR SALE OR DISTRIBUTION the location at which it forms?” and “What ensures that the daughter chromosomes lie on opposite sides of it?”

© Jones & Bartlett Learning, LLC Figure 9.6 ©E. coliJones generate & anucleateBartlett cells Learning, when chromosome LLC 9.4 Mutations in Division or segregation fails. Cells with chromosomes stain blue; daughter NOT FOR SALE OR DISTRIBUTION cells lackingNOT chromosomes FOR SALEhave no blueOR stain. DISTRIBUTION This field shows Segregation Affect Cell Shape cells of the mukB mutant; both normal and abnormal divisions can be seen. Photo courtesy of Sota Hiraga, Kyoto University. Key Concepts

© Jones &• Bartlettfts mutants Learning, form long filaments LLC because the septum that © Jones & Bartlett Learning, LLC NOT FOR SALEdivides OR the daughter DISTRIBUTION bacteria fails to form. NOT FOR SALEThe bacteria OR DISTRIBUTIONcontinue to grow—and even continue • Minicells form in mutants that produce too many septa; to segregate their daughter chromosomes—but they are small and lack DNA. septa do not form. Thus, the cell consists of a very • Anucleate cells of normal size are generated by partition long filamentous structure, with the nucleoids (bac- mutants, in which the duplicate chromosomes fail to terial chromosomes) regularly distributed along the separate. © Jones & Bartlett Learning, LLC length of the cell. This© phenotypeJones & is displayedBartlett by Learning, fts LLC NOT FOR SALE OR DISTRIBUTION mutants (named for temperature-sensitiveNOT FOR SALE filamen OR DISTRIBUTION- A difficulty in isolating mutants that affect cell division is tation), which identify a defect or multiple defects that mutations in the critical functions might be lethal and/ that lie in the division process itself. or pleiotropic. Most mutations in the division apparatus • Minicells form when septum formation occurs too have been identified as conditional mutants (whose division frequently or in the wrong place, with the result that is affected© Jones under &nonpermissive Bartlett Learning, conditions; typically,LLC they one© ofJones the new & daughter Bartlett cells Learning, lacks a chromosome. LLC are temperatureNOT FOR sensitive). SALE ORMutations DISTRIBUTION that affect cell divi- TheNOT minicell FOR has aSALE rather small OR size DISTRIBUTION and lacks DNA, but sion or chromosome segregation cause striking phenotypic otherwise appears morphologically normal. Anucleate changes. Figure 9.5 and Figure 9.6 illustrate the opposite cells form when segregation is aberrant; like minicells, consequences of failure in the division process and failure they lack a chromosome, but because septum forma- in segregation: tion is normal, their size is unaltered. This phenotype © Jones & Bartlett• Long Learning,filaments form LLC when septum formation is© Jones & Bartlettis caused by Learning, par (partition) LLC mutants (named because NOT FOR SALEinhibited, OR DISTRIBUTION but chromosome replication is unaffected.NOT FOR SALEthey are OR defective DISTRIBUTION in chromosome segregation).

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are all transmembrane proteins. The final structure is some- 9.5 FtsZ Is Necessary times called the septal ring. It consists of a multiprotein © Jones for& Bartlett Septum Learning, Formation LLC © Jonescomplex & Bartlettthat is presumed Learning, to have theLLC ability to constrict the NOT FOR SALE OR DISTRIBUTION NOT membrane.FOR SALE One OR of the DISTRIBUTION last components to be incorporated into the septal ring is FtsW, which is a protein belonging to Key Concepts the SEDS family. The ftsW gene is expressed as part of an operon with ftsI, which encodes a transpeptidase (also called • The product of ftsZ is required for septum formation. PBP3 for penicillin-binding protein 3), a membrane-bound • FtsZ is a GTPase that© resembles Jones tubulin, & Bartlett and polymerizes Learning, LLCprotein that has its catalytic ©site Jones in the periplasm. & Bartlett FtsW Learning, is LLC to form a ring on the inside of the bacterial envelope. It responsible for incorporating FtsI into the septal ring. This is required to recruit theNOT enzymes FOR needed SALE to form OR the DISTRIBUTION NOT FOR SALE OR DISTRIBUTION septum. suggests a model for septum formation in which the transpeptidase activity then causes the peptidoglycan to grow The gene ftsZ plays a central role in division. Mutations in inward, thus pushing the inner membrane and pulling the ftsZ block septum formation and generate filaments. Over- outer membrane. expression© Jones induces & minicells Bartlett by Learning,causing an increased LLC num- © Jones & Bartlett Learning, LLC ber ofNOT septation FOR events SALE per unit OR cell DISTRIBUTION mass. FtsZ (the protein) NOT FOR SALE OR DISTRIBUTION recruits a battery of cell division proteins that are responsible 9.6 min and noc/slm Genes for synthesis of the new septum. Regulate the Location of FtsZ functions at an early stage of septum formation. the Septum © Jones &Early Bartlett in the division Learning, cycle, FtsZ LLC is localized throughout the© Jones & Bartlett Learning, LLC cytoplasm, but prior to cell division FtsZ becomes localized in NOT FORa ringSALE around OR the DISTRIBUTION circumference at the mid-cell position. TheNOT FOR SALE OR DISTRIBUTION Key Concepts structure is called the Z-ring, which is shown in Figure 9.7. The formation of the Z-ring is the rate-limiting step in septum • The location of the septum is controlled by minC, -D, and formation, and its assembly defines the position of the septum. -E, and by noc/slmA. In a typical division cycle,© Jonesit forms in & the Bartlett center of Learning, the cell 1 LLC• The number and location of© septa Jones are determined & Bartlett by the Learning, LLC to 5 minutes after division, remains for 15 minutes, and then ratio of MinE/MinCD. quickly constricts to pinchNOT the cellFOR into SALE two. OR DISTRIBUTION• Dynamic movement of the MinNOT proteins FOR in theSALE cell sets OR up DISTRIBUTION The structure of FtsZ resembles tubulin, suggesting that a pattern in which inhibition of Z-ring assembly is highest assembly of the ring could resemble the formation of micro- at the poles and lowest at mid-cell. tubules in eukaryotic cells. FtsZ has GTPase activity, and • SlmA/Noc proteins prevent septation from occurring in the space occupied by the bacterial chromosome. GTP ©cleavage Jones is used & Bartlett to support Learning, the oligomerization LLC of FtsZ © Jones & Bartlett Learning, LLC monomers into the ring structure. The Z-ring is a dynamic structure,NOT in FORwhich thereSALE is continuous OR DISTRIBUTION exchange of subunits Clues to theNOT localization FOR of SALE the septum OR were DISTRIBUTION first provided by with a cytoplasmic pool. minicell mutants. The original minicell mutation lies in the Two other proteins needed for division, ZipA and FtsA, locus minB. Deletion of minB generates minicells by allow- interact directly and independently with FtsZ. ZipA is an ing septation to occur near the poles instead of at mid-cell, integral membrane protein that is located in the inner bac- and therefore the role of the wild-type minB locus is to sup- © Jones &terial Bartlett membrane. Learning, It provides LLC the means for linking FtsZ to© Jonespress septation& Bartlett at the Learning, poles. The minB LLC locus consists of three NOT FORthe SALE membrane. OR FtsADISTRIBUTION is a cytosolic protein, but is often foundNOT genes,FOR minC,SALE -D, OR and DISTRIBUTION-E. The products of minC and minD associated with the membrane. The Z-ring can form in the form a division inhibitor. MinD is required to activate MinC, absence of either ZipA or FtsA, but it cannot form if both are which prevents FtsZ from polymerizing into the Z-ring. absent. Both are needed for subsequent steps. This suggests Expression of MinCD in the absence of MinE, or over- that they have overlapping roles in stabilizing the Z-ring and expression even in the presence of MinE, causes a general- perhaps in linking it to the© Jonesmembrane. & Bartlett Learning, LLCized inhibition of division. The© Jonesresulting &cells Bartlett grow as longLearning, LLC The products of severalNOT other FOR fts SALEgenes join OR the DISTRIBUTION Z-ring filaments without septa. ExpressionNOT of FOR MinE SALE at levels compaOR DISTRIBUTION- in a defined order after FtsA has been incorporated. They rable to MinCD confines the inhibition to the polar regions, thus restoring normal growth. The determinant of septation at the proper (mid-cell) site is, therefore, the ratio of MinCD to MinE. © Jones & Bartlett Learning, LLC The localization© Jones activities & Bartlett of the Min Learning, system are LLCdue to a NOT FOR SALE OR DISTRIBUTION remarkableNOT dynamic FOR behavior SALE of MinDOR DISTRIBUTION and MinE, which is illustrated in Figure 9.8. MinD, an ATPase, oscillates from one end of the cell to the other on a rapid time scale. MinD- ATP binds to and accumulates at the bacterial lipid mem- Figure 9.7 Immunofluorescence with an antibody against FtsZ brane at one pole of the cell, is released, and then rebinds to © Jones &shows Bartlett that it is localizedLearning, at the mid-cell.LLC © Jonesthe opposite & Bartlett pole. The Learning, periodicity ofLLC this process takes about NOT FORPhoto SALE courtesy of William OR Margolin, DISTRIBUTION University of Texas Medical School at Houston. NOT 30FOR seconds, SALE so thatOR multiple DISTRIBUTION oscillations occur within one

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Oscillation of Min proteins in E. coli nucleoid-free spaces at the poles and mid-cell. The combina- tion of nucleoid occlusion and the Min system promotes the © Jones & Bartlett Learning, LLC MinE © JonesZ-rings & toBartlett form, and Learning, thus cell division LLC to occur, at mid-cell. NOT FOR SALE OR DISTRIBUTION MinD NOT FOR SALE OR DISTRIBUTION MinC 9.7 Partition Involves Separation MinD binds to membrane at one pole MinC binds to MinD of the Chromosomes A MinE ring forms at the edge of MinD © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONKey Concepts NOT FOR SALE OR DISTRIBUTION

• Daughter chromosomes are disentangled from each other by topoisomerases. • Chromosome segregation occurs concurrently with DNA MinE ring promotes dissociation replication; that is, it begins before DNA replication is © Jones &of MinDBartlett from the Learning, membrane LLC finished.© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION • CondensationNOT of FOR the chromosome SALE OR by MukBEF DISTRIBUTION or SMC proteins is necessary for proper chromosome orientation and segregation.

Partition is the process by which the two daughter chromo- © Jones & Bartlett Learning, LLC © Jonessomes & find Bartlett themselves Learning, on either side LLC of the position at which MinD diffuses and binds to opposite pole NOT FOR SALE OR DISTRIBUTIONMinE ring dissociates and reforms NOT theFOR septum SALE forms. OR Two DISTRIBUTION types of event are required for proper at edge of new MinD zone partition: • The two daughter chromosomes must be released from one another so that they can segregate following termination. This requires disentangling of DNA © Jones & Bartlett Learning, LLC regions that are coiled© around Jones each & other Bartlett in the vicin Learning,- LLC NOT FOR SALE OR DISTRIBUTION ity of the terminus.NOT Mutations FOR affecting SALE partition OR DISTRIBUTION Figure 9.8 MinCD is a division inhibitor whose action is map in genes coding for topoisomerases—enzymes confined to the polar sites by MinE. with the ability to pass DNA strands through one another. The mutations prevent the daughter chro- bacterial cell generation. MinC, which cannot move on its mosomes from segregating, with the result that the own, ©oscillates Jones as a& passenger Bartlett protein Learning, bound to LLC MinD. MinE DNA© Jones is located & in Bartlett a single, largeLearning, mass at mid-cell. LLC formsNOT a ring FORaround SALE the cell atOR the DISTRIBUTIONedge of the zone of MinD. SeptumNOT formation FOR SALE then releases OR DISTRIBUTION an anucleate cell and The MinE ring moves toward MinD at the poles and is nec- a cell containing both daughter chromosomes. This essary for ATP hydrolysis and the release of MinD from the tells us that the bacterium must be able to disentan- membrane. The MinE ring then disassembles and reforms at gle its chromosomes topologically in order to be able the edge of the MinD zone that forms at the opposite pole. to segregate them into different daughter cells. © Jones &MinD Bartlett and MinE Learning, are each required LLC for the dynamics of the© Jones &• BartlettThe two Learning,daughter chromosomes LLC must move NOT FORother. SALE The ORconsequence DISTRIBUTION of this dynamic behavior is that theNOT FOR SALEapart during OR DISTRIBUTION partition. The original models for concentration of the MinC inhibitor is lowest at mid-cell and chromosome segregation suggested that the cell highest at the poles, which directs FtsZ assembly at mid-cell envelope grows by insertion of material between and inhibits its assembly at the poles. membrane-attachment sites of the two chromo- Another process, called nucleoid occlusion, prevents somes, thus pushing them apart. In fact, the cell Z-ring formation over ©the Jones bacterial & chromosome Bartlett Learning,and thus LLC wall and membrane© growJones heterogeneously & Bartlett overLearning, LLC prevents the septum fromNOT bisecting FOR anSALE individual OR chromoDISTRIBUTION- the whole cell surface.NOT Current FOR models SALE of bacterial OR DISTRIBUTION some at cell division. A protein called SlmA, which interacts chromosome segregation do not require attach- with FtsZ, is necessary for nucleoid occlusion in E. coli. SlmA ment to the membrane, although the confinement binds specifically to at least 24 sites on the bacterial chromo- that is provided by the membrane is thought to be some. DNA binding activates SlmA to antagonize the poly- necessary to help push chromosomes apart. Some merization© Jones of FtsZ, & which Bartlett prevents Learning, septum formation LLC in this of© the Jones machinery & Bartlettand forces Learning,that drive segregation LLC regionNOT of the FOR cell. In SALE Bacillus ORsubtilis, DISTRIBUTION a DNA-binding protein haveNOT been FOR identified SALE but ORthe picture DISTRIBUTION is still incom- called Noc performs a similar nucleoid occlusion role, but plete. The first important step is to promote sepa- by a different mechanism. Noc interacts directly with the ration of the newly replicated origin regions of the membrane, rather than with FtsZ, and this interaction inter- chromosome. As new origins move to new cellular feres with the assembly of the cell division machinery. The locations (Figure 9.4), the rest of the chromosomes © Jones &bacterial Bartlett nucleoid Learning, takes up a LLClarge volume of the cell, and as© Jones & Bartlettfollow after Learning, they are replicated. LLC The replicated chro- NOT FORa SALEresult this OR process DISTRIBUTION restricts Z-ring assembly to the limitedNOT FOR SALEmosomes OR are DISTRIBUTION capable of abrupt movements, which

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indicates that some regions are held together for an those necessary for partition of low-copy-number plasmids. interval of time before they rapidly separate. The These discoveries and analyses in current research will © Jones & Bartlettfinal stepLearning, is to separate LLC newly replicated terminus© Joneslead to& aBartlett better understanding Learning, of LLC how genomes are posi- NOT FOR SALEregions OR DISTRIBUTION of the chromosome. NOT tionedFOR inSALE the cell. OR DISTRIBUTION Mutations that affect the partition process itself are rare. Segregation is interrupted by mutations of the muk class in E. coli, which give rise to anucleate progeny at a much increased 9.8 Chromosomal Segregation frequency: Both daughter chromosomes remain on the same Might Require Site-Specific side of the septum instead© Jones of segregating. & Bartlett Mutations Learning, in the LLC © Jones & Bartlett Learning, LLC muk genes are not lethal,NOT and FORthey identify SALE components OR DISTRIBUTION of RecombinationNOT FOR SALE OR DISTRIBUTION the apparatus that segregate the chromosomes. The gene mukB encodes a large (180-kD) protein, which has the same Key Concepts general type of organization as the two groups of structural maintenance of chromosomes (SMC) proteins that are • The Xer site-specific recombination system acts on a involved© Jones in condensing & Bartlett and in holdingLearning, together LLC eukaryotic target sequence© Jones near & the Bartlett chromosome Learning, terminus to LLC chromosomes.NOT FOR SMC-like SALE proteins OR DISTRIBUTION have also been found in recreateNOT monomers FOR if a SALEgeneralized OR recombination DISTRIBUTION event other bacteria and mutations in their genes also increase the has converted the bacterial chromosome to a dimer. frequency of anucleate cells. Another phenotype of mukB • FtsK acts at the terminus of replication to promote the final mutants is that the organization of the chromosome is altered separation of chromosomes and their transport through the growing septum. from that shown in Figure 9.4; origins and termini are reori- © Jones &ented Bartlett toward Learning,the poles for theLLC entire cell cycle. Therefore,© Jones & Bartlett Learning, LLC NOT FORMukB SALE also OR acts DISTRIBUTIONto properly orient and position the originNOT AfterFOR replication SALE OR has DISTRIBUTIONcreated duplicate copies of a bacte- regions of the chromosome during segregation. rial chromosome or plasmid, the copies can recombine. Initial insight into the role of MukB was the discovery Figure 9.10 demonstrates the consequences. A single inter- that some mutations in mukB can be suppressed by muta- molecular recombination event between two circles generates tions in topA, the gene that encodes topoisomerase I. MukB a dimeric circle; further recombination can generate higher forms a complex with two© Jonesother proteins, & Bartlett MukE and Learning, MukF, LLCmultimeric forms. Such an ©event Jones reduces & theBartlett number Learning, of LLC and the MukBEF complexNOT is consideredFOR SALE to be OR a condensin DISTRIBUTION physically segregating units. InNOT the extreme FOR SALEcase of aOR single- DISTRIBUTION analogous to eukaryotic condensins. A defect in this function copy plasmid that has just replicated, formation of a dimer by can be compensated for by preventing topoisomerases from recombination means that the cell only has one unit to segre- relaxing negative supercoils; the resulting increase in super- gate, and the plasmid therefore must inevitably be lost from coil density helps to restore the proper state of condensation one daughter cell. To counteract this effect, plasmids often and allow© Jones segregation. & Bartlett Figure 9.9 Learning, shows one modelLLC for the have site-specific© Jones recombination & Bartlett systems Learning, that act upon LLC par- role ofNOT condensation. FOR SALE The parental OR DISTRIBUTION genome is centrally posi- ticular sequencesNOT to FOR sponsor SALE an intramolecular OR DISTRIBUTION recombination tioned. It must be decondensed in order to pass through the that restores the monomeric condition. For example, plasmid replication apparatus. The daughter chromosomes emerge P1 encodes the Cre protein-lox site recombination system from replication, are disentangled by topoisomerases, and for this purpose. Scientists have further exploited the Cre-lox then passed in an uncondensed state to MukBEF, which system extensively for genetic engineering in many different © Jones &causes Bartlett them to Learning, form condensed LLC masses at the positions that© Jones & Bartlett Learning, LLC NOT FORwill SALE become OR the DISTRIBUTIONcenters of the daughter cells. NOT FOR SALE OR DISTRIBUTION It is likely that MukBEF (or SMC in other bacteria) Monomeric circles works with other factors to promote the initial steps in segregation of the origin region of the chromosome. Research- ers have identified some of these factors in other bacteria, Recombination such as partition genes, ©called Jones parA &and Bartlett parB, that Learning, resemble LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Parental nucleoid Condensed Condensed Dimeric circle daughter daughter © Jones & Bartlett Learning, LLC © Jones & BartlettRecombination Learning, LLC NOT FOR SALEReplication OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Monomeric circles MukB

Figure 9.9 The DNA of a single parental nucleoid becomes Figure 9.10 Intermolecular recombination merges monomers © Jones &decondensed Bartlett during Learning, replication. LLC MukB is an essential component© Jonesinto dimers, & Bartlett and intramolecular Learning, recombination LLC releases individual NOT FORof SALE the apparatus OR thatDISTRIBUTION recondenses the daughter nucleoids. NOT unitsFOR from SALE oligomers. OR DISTRIBUTION

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organisms. These systems are also discussed in the chapter titled Homologous and Site-Specific Recombination. © Jones & BartlettThe same Learning,type of events LLC can occur with the bacterial© Jones & Bartlett Learning, LLC NOT FORchromosome; SALE OR Figu DISTRIBUTIONre 9.11 shows how such an event affects itsNOT FOR SALE OR DISTRIBUTION segregation. If no recombination occurs, there is no problem, and the separate daughter chromosomes can segregate to the Chromosomes are linked daughter cells. A dimer will be produced, however, if homol- at recombination site ogous recombination occurs between the daughter chromosomes produced by a replication© Jones cycle. & IfBartlett there has beenLearning, such LLC © Jones & Bartlett Learning, LLC dif a recombination event,NOT the daughter FOR SALEchromosomes OR DISTRIBUTION cannot NOT FOR SALE OR DISTRIBUTION separate. In this case, a second recombination is required to achieve resolution in the same way as a plasmid dimer. Most bacteria with circular chromosomes possess the Xer site-specific recombination system. In E. coli, this con- Xer recombinase sists of© twoJones recombinases, & Bartlett XerC Learning, and XerD, which LLC act on a forms© Jonesjunction at &dif Bartlett Learning, LLC 28-base-pairNOT FOR(bp) target SALE site ORcalled DISTRIBUTION dif that is located in the NOT FOR SALE ORdif DISTRIBUTION terminus region of the chromosome. The use of the Xer system is related to cell division in an interesting way. The rele- vant events are summarized in Figure 9.12. XerC can bind to a pair of dif sequences and form a Holliday junction between © Jones &them. Bartlett The complex Learning, might formLLC soon after the replication© Jones & BartlettFtsK is required Learning, for resolution LLC NOT FORfork SALE passes OR over DISTRIBUTION the dif sequence, which explains how theNOT FOR SALE OR DISTRIBUTION two copies of the target sequence can find each other consis- tently. Resolution of the junction to give recombinants, however, occurs only in the presence of FtsK, a protein located in the septum that is required for chromosome segregation © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Cell has one circular chromosome Figure 9.12 A recombination event creates two linked NOT FOR SALE OR DISTRIBUTIONchromosomes. Xer creates a HollidayNOT junction FOR at SALE the dif site, OR but DISTRIBUTION can resolve it only in the presence of FtsK.

Bidirectional replication begins and cell division. In addition, the dif target sequence must be located in a region of approximately 30 kb; if it is moved out- © Jones & Bartlett Learning, LLC side of this© region, Jones it cannot & Bartlett support theLearning, reaction. Remem LLC - NOTReplication FOR SALEresults in chromosomeOR DISTRIBUTION movement ber that theNOT terminus FOR region SALE of the OR chromosome DISTRIBUTION is located near the septum prior to cell division as discussed in the section The Shape and Spatial Organization of a Bacterium Are Important During Chromosome Segregation and Cell Division earlier in this chapter. © Jones & Bartlett Learning, LLC © JonesThe & Bartlettbacterium, however,Learning, should LLC have site-specific recom- NOT FOR SALENo OR recombination DISTRIBUTION General recombination NOT binationFOR SALE at dif only OR when DISTRIBUTION there has already been a general Daughter recombination event to generate a dimer. (Otherwise, the chromosomes Movement is move apart constrained site-specific recombination would create the dimer!) How does the system know whether the daughter chromosomes exist as independent monomers or have been recombined © Jones & Bartlett Learning, LLCinto a dimer? One answer is the© Jonestiming of &chromosome Bartlett segLearning,- LLC Daughter Site-specific chromosomes NOT recombinationFOR SALE OR DISTRIBUTIONregation. Remember that the terminusNOT FOR is the SALElast region OR of the DISTRIBUTION segregate releases chromosomes chromosome to be segregated. If there has been no recombi- X nation, the two chromosomes move apart from one another shortly after they are replicated. The ability to move apart from Daughter chromosomes one another, however, will be constrained if a dimer has been © Jones & Bartlettseg Learning,regate LLC formed. This© forces Jones the terminus& Bartlett region Learning, to remain in the LLC vicin- NOT FOR SALE OR DISTRIBUTION ity of the septum,NOT whereFOR sites SALE are exposed OR DISTRIBUTION to the Xer system. Another factor that promotes separation of the terminus is the FtsK protein. Bacteria that have the Xer system Figure 9.11 A circular chromosome replicates to produce always have an FtsK homolog, and vice versa, which suggests two monomeric daughters that segregate to daughter cells. A that the system has evolved so that resolution is connected generalized recombination event, however, generates a single © Jones &dimeric Bartlett molecule. Learning, This can be resolvedLLC into two monomers by a © Jonesto the & septum. Bartlett FtsK Learning, is a large transmembrane LLC protein. Its NOT FORsite-specific SALE OR recombination. DISTRIBUTION NOT N-terminalFOR SALE domain OR isDISTRIBUTION associated with the membrane and

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causes it to be localized to the septum. Its C-terminal domain examine Epidermal Growth Factor (EGF) and its receptor, has two functions. One is to cause Xer to resolve a dimer into EGFR—a member of the erbB family of four related recep- © Jones two& Bartlett monomers. Learning, It also has an LLC ATPase activity, which it uses© Jonestors. These& Bartlett two proteins, Learning, EGF and LLCEGFR, and the genes that NOT FORto SALE pump DNA OR through DISTRIBUTION the septum. NOT encodeFOR SALEthem are OR the firstDISTRIBUTION two elements in the pathway. EGF A special type of chromosome segregation occurs during is a peptide hormone (as opposed to a steroid hormone such sporulation in B. subtilis. One daughter chromosome must be as estrogen). The EGFR specifically binds EGF in a lock- segregated into the forespore compartment. This is an unusual and-key type of mechanism. EGFR is a one-pass membrane process that involves transfer of the chromosome across the protein in the family known as receptor tyrosine kinases nascent septum. One of© theJones sporulation & Bartlett genes, spoIIIE,Learning, is LLC(RTK), as shown in Figure 9.13© aJones. The receptor & Bartlett has an exter Learning,- LLC required for this process.NOT The SpoIIIE FOR proteinSALE resembles OR DISTRIBUTION FtsK, nal domain (that is outside theNOT cell) FORthat binds SALE EGF, ORa single DISTRIBUTION is located at the septum, and has a translocation function that membrane-spanning domain, and an internal cytoplasmic pumps DNA through to the forespore compartment. domain with intrinsic tyrosine kinase activity. The local membrane composition (e.g., cholesterol) can modulate the dynamics of the signal transduction pathway. 9.9© T Joneshe E ukary& Bartlettotic Learning, Growth LLC Hormone© Jonesbinding & toBartlett receptor Learning, stabilizes receptorLLC FactorNOT FORSignal SALE T ORransduction DISTRIBUTION dimerizationNOT (usually FOR homodimerization, SALE OR DISTRIBUTION but heterodimers with other erbB family members can occur), which leads Pathway Promotes Entry to multiple cross-phosphorylation events of each receptor’s to S Phase cytoplasmic domain. The only function of the hormone is to stabilize receptor dimerization. Each receptor phosphory- © Jones & Bartlett Learning, LLC © Joneslates the& Bartlettother on a Learning,set of five tyrosine LLC amino acid residues NOT FOR SALEKey OR Conc DISTRIBUTIONepts NOT inFOR the cytoplasmic SALE OR domain, DISTRIBUTION as shown in FIGURE 9.13b. Each phosphorylated tyrosine (Tyr-P) serves as a docking site for • The function of a growth factor is to stabilize dimerization a specific adaptor protein to bind to the receptor, as shown of its receptor and subsequent phosphorylation of the in FIGURE 9.13c. We will examine a single pathway, but it is cytoplasmic domain of the receptor. important to keep in mind that cells contain many different • The function of the growth© Jones factor receptor & Bartlett is to recruit Learning, the LLCreceptors that are active at the© same Jones time, &and Bartlett each receptor Learning, LLC exchange factor SOSNOT to the membraneFOR SALE to activate OR RAS. DISTRIBUTIONhas multiple docking sites forNOT multiple FOR proteins. SALE The OR reality DISTRIBUTION • The function of activated RAS is to recruit RAF to the is that it is not a pathway but rather an information network. membrane to become activated. Paradoxically, hormone binding to the receptor also • The function of RAF is to initiate a phosphorylation cascade leading to the phosphorylation of a set of causes clathrin-mediated endocytosis of the hormone recep- transcription factors that can enter the nucleus and tor complex to the lysosomal complex, where it is targeted for begin© Jones S phase. & Bartlett Learning, LLC destruction,© and Jones thus turnover. & Bartlett This traffickingLearning, is regulated LLC NOT FOR SALE OR DISTRIBUTION by microtubuleNOT deacetylation, FOR SALE which OR controls DISTRIBUTION the proportion The vast majority of eukaryotic cells in a multicellular indi- of receptors that are returned to the surface. This is part of vidual are not growing; that is, they are in the cell cycle stage an important attenuation mechanism to prevent accidental of G0, as we saw in the beginning of this chapter. Stem cells triggering of the pathway and it means that growth factor and most embryonic cells, however, are actively growing. A must be continually present to propagate a sustained signal. © Jones &growing Bartlett cell exiting Learning, mitosis has LLC two choices—it can enter G1© JonesThe & Bartlettthird member Learning, of the signal LLC transduction pathway is NOT FORand SALE begin aOR new DISTRIBUTION round of cell division or it can stop dividingNOT theFOR RAS SALE protein OR(encoded DISTRIBUTION by the ras gene). RAS is a member and enter G0, a quiescent stage and, if so programmed, begin of a large family of G-proteins, proteins that bind a guanosine differentiation. This decision is controlled by the develop- nucleotide, either GTP (for the active form of RAS) or GDP mental history of the cell and the presence or absence of (for the inactive form). RAS is connected to the membrane by growth factors and their receptors. a prenylated (lipid) tail, and typically found in nanoclusters For a cell to begin ©the Jones cell cycle & from Bartlett G0, or Learning, continue LLCon the cytoplasmic side of the© membrane Jones &to enhanceBartlett down Learning,- LLC to divide after M phase,NOT it must FOR be programmed SALE OR to DISTRIBUTIONexpress stream signaling. To continue theNOT flow FOR of information SALE throughOR DISTRIBUTION the proper growth factor receptor gene. Elsewhere in the the signal transduction pathway communicating that a growth organism, typically in a master gland (but can also occur factor is present, inactive RAS must be converted from RAS- in neighboring cells), the gene for the proper growth factor GDP to RAS-GTP by a protein called Son of Sevenless (SOS), must be expressed. The signal transduction pathway is the a guanosine nucleotide exchange factor (GEF) that exchanges biochemical© Jones mechanism & Bartlett by which Learning, the growth factor LLC signal to GTP for GDP.© Jones Its function & Bartlett is to remove Learning, the GDP from LLC RAS growNOT is communicated FOR SALE from ORits source DISTRIBUTION outside of the cell into and replaceNOT it with FOR GTP, asSALE shown ORin FIGU DISTRIBUTIONRE 9.13d. RAS also the nucleus to ultimately cause that cell to begin replication has a weak intrinsic phosphatase (GTPase) activity that slowly and growth. The pathway that we describe in this section is converts GTP to GDP. Again, this provides a mechanism to universal in eukaryotes, ranging from yeast to humans. ensure that growth factor must be present continually for the The genes that encode elements of the signal trans- signal to propagate. © Jones &duction Bartlett pathway Learning, are proto-oncogenes LLC , genes that when© JonesTo & activate Bartlett RAS, Learning, SOS must be LLC specifically recruited to NOT FORaltered SALE can OR cause DISTRIBUTION cancer. As an example of this pathway, weNOT theFOR membrane SALE in OR order DISTRIBUTION to interact with RAS-GDP. It is the

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GF (b) (a) GF © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION GF

RTK RTK RTK © Jones & Bartlett Learning, LLC © JonesP & Bartlett Learning, LLC P SOS RTK NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Cytoplasm Grb2

RAS GDP RAS GTP SOS SOS Inactive © Jones & Bartlett Learning, LLCRTK © Jones & Bartlett Learning,RTK LLC Active RTK RTK NOT FOR SALE OR DISTRIBUTIONP NOT FOR SALE OR DISTRIBUTIONP P P

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Figure 9.13 The signal transductionNOT FOR pathway. SALE (a) ORGrowth DISTRIBUTION factors and growth factor receptors: The growthNOT factor FOR extracellular SALE domain OR DISTRIBUTION will bind the growth factor in a lock-and-key fashion. The growth factor receptor intracellular domain contains an intrinsic protein kinase domain called RTK. (b) Growth factor binding to its receptor will stabilize receptor dimerization, leading to phosphorylation of each cytoplasmic domain on tyrosine. The phosphotyrosine residues can serve as binding sites for proteins such as Grb2, shown here. (c) Grb2 binds the Tyr-P so that its binding partner SOS, a guanosine nucleotide exchange factor, is brought to the membrane and can activate the inactive RAS-GDP. (d) SOS removes the GDP, replacing it with GTP, activating RAS. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC membraneNOT phospholipidsFOR SALE themselves OR DISTRIBUTION that serve to unlock activated: aNOT structurally FOR inactive SALE form OR of DISTRIBUTIONRAF (also known as an auto-inhibitory domain so that SOS can bind to RAS. MAPKKK or mitogen-activated protein kinase kinase kinase), SOS is in a complex with an adaptor protein called Grb2, an a serine/threonine protein kinase. The activation of RAF on interesting protein with two domains: an SH2 domain that the membrane has been one of the most baffling steps, with binds Tyr-P, and an SH3 domain that binds proteins contain- researchers having proposed many models over the years. The © Jones &ing Bartlett another SH3 Learning, domain. The LLC specificity for binding to the© Jonesonly function & Bartlett of RAS-GTP Learning, is to recruit LLC RAF to the membrane NOT FORreceptor SALE lies OR in theDISTRIBUTION amino acids surrounding each Tyr-P. TheNOT forFOR activation; SALE it ORdoes nothingDISTRIBUTION else. The most recent model is only function of the growth factor is to stabilize dimerization of the dimer model for RAS-mediated activation of a dimer of the receptor, which leads to its phosphorylation, which in turn RAF (see Figure 9.14). This activation is facilitated by the leads to recruitment of SOS to the membrane to activate RAS. fact that RAS is present in the membrane in high concentra- Inactive RAS-GDP and active RAS-GTP are in a dynamic tion in nanoclusters. This high concentration of RAS leads to equilibrium controlled ©by Jones the exchange & Bartlett factor GEFLearning, and LLCthe formation of a dimer of RAS-GTP© Jones which & Bartlett facilitates Learning,the LLC another set of proteins thatNOT stimulate FOR theSALE intrinsic OR GTPase DISTRIBUTION of next step. RAF activation onNOT the membraneFOR SALE involves OR itsDISTRIBUTION RAS, such as RAS GAP (GTPase activating protein). dimerization leading to the RAS-assisted unfolding of the ras oncogenic mutations that constitutively activate RAS autoinhibitory domains of the RAF dimer. This then allows are among the most frequent oncogenic mutations found in phosphorylation by another membrane associated kinase, tumors. The most common mutation is a single nucleotide SRC, and release of the RAF dimer from the platform. change© thatJones causes & a Bartlett single amino Learning, acid change, LLC resulting in Activated© JonesRAF phosphorylates & Bartlett a secondLearning, kinase, LLCsuch as alteredNOT function. FOR RAS SALEONC has OR a key DISTRIBUTION altered property: It binds one of the NOTmitogen-activated FOR SALE kinase OR (MEK) DISTRIBUTION factors, which GTP with a higher affinity than GDP. The consequence is then phosphorylates a third kinase, such as one of the extra- that it no longer requires a growth factor to trigger activation; cellular signal-regulated kinase (ERK) factors, which can then it is constitutively active. This kind of mutation is referred to phosphorylate and activate the set of transcription factors as a dominant gain-of-function mutation. such as MYC, JUN, and FOS. This allows their entry into the © Jones & BartlettActivated Learning,RAS, RAS-GTP, LLC now itself serves as a dock-© Jonesnucleus & toBartlett begin transcribing Learning, the genes LLC to prepare for transit NOT FORing SALE site to ORrecruit DISTRIBUTION the fourth member of the pathway to beNOT throughFOR SALE G1 and ORentry DISTRIBUTION into S phase. Again, note that this is

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Ras in nanodusters membrane membrane membrane © Jones & Bartlett Learning,HVR/CAAX LLC © JonesHVR/CAAX & Bartlett Learning, LLC HVR/CAAX GAP Binding NOT FOR SALERas OR DISTRIBUTIONRas Ras NOTRa FORs SALE OR DISTRIBUTIONRas Ras GDP GDP GTP GTP GTP GTP GEF / Unbinding RBD RBD Mutation RAF RAF RBD RBD RAF RBD RAF RBD RAF RAF KD KD KD KD KD KD © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC dimerization dimerization dimerization dissociation dissociation dissociation /clustering NOT FOR SALE OR DISTRIBUTION/clustering NOT/clustering FOR SALE OR DISTRIBUTION

membrane membrane membrane HVR/CAAX HVR/CAAX HVR/CAAX GAP Binding Ras Ras Ras Ras Ras Ras © JonesGDP GDP & Bartlett Learning, LLC GTP GTP © Jones & Bartlett GTPLearning,GTP LLC GEF / Unbinding RBD RBD NOT FOR SALE OR MutationDISTRIBUTION NOT FOR SALE OR DISTRIBUTION RAF RAF RBD RBD RAF RBD RAF RBD RAF RAF KD KD KD KD KD KD

MEK/Erk © Jones &Figu Bartlettre 9.14 Dimer Learning, model for RLLCas-mediated activation of Raf. R©as Jones-GTP forms & dimers Bartlett to cooperatively Learning, activate LLC Raf. NOT FORa SALEdescription OR of DISTRIBUTIONa single pathway within a network that hasNOT andFOR if theySALE are ORmutated, DISTRIBUTION premature cell cycle progression extensive crosstalk between members. In addition, this kinase results in wee, tiny cells.) This also means that the balance of cascade is modulated by an extensive network of phosphatases. kinases and phosphatases regulates the activity of the CDKs. We will focus on the G1 to S phase transition. (There is sim- © Jones & Bartlett Learning, LLCilar tight control at the G2 to© M Jones transition & and Bartlett within variLearning,- LLC 9.10 Checkpoint Control for ous stages of mitosis and meiosis.) The signal for entry into NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Entry into S Phase: p53, a S phase is a positive signal controlled by negative regulators. The S to G2 transition occurs when replication is completed. Guardian of the Checkpoint For a cell to be allowed to progress from G1 to S phase, two major requirements must be met. The cell must have grown ©Key Jones Conc &epts Bartlett Learning, LLC a specific amount© Jones in size & andBartlett there must Learning, be no DNA LLC damage. The worst thing that a cell can do is to replicate damaged NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION • The tumor suppressor proteins p53 and Rb act as DNA. To ensure that both requirements are met, the CDK/ guardians of cell integrity. cyclin complexes are controlled by checkpoint proteins. Two • A set of ser/thr protein kinases called cyclin-dependent of the most important are the transcription factors p53 and Rb. kinases control cell cycle progression. These two proteins are in a class called tumor suppressor pro- • Cyclin proteins are required to activate cyclin-dependent teins. As guardians of the cell cycle, these proteins ensure that © Jones & Bartlettkinase proteins. Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE• Inhibitor OR proteins DISTRIBUTION negatively regulate the cyclin/cyclin- NOT FOR SALE OR DISTRIBUTION dependent kinases. Cyclin ϩ CDK • Activator proteins called CDK-activating kinases positively regulate the cyclin/cyclin-dependent kinases. Inactive

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Progression through the cell cycle, after the initial activation CAK by growth factor, requiresNOT continuous FOR growthSALE factor OR presenceDISTRIBUTION NOT FOR SALE OR DISTRIBUTION and is tightly controlled by a second set of ser/thr protein kinases called cyclin-dependent kinases (CDKs; and some- CKI times cell division–dependent kinases). The CDKs themselves are controlled in a very complex fashion as shown in © Jones & Bartlett Learning, LLC © Jones & BartlettCdc25 Learning,Chk 1ϩ2 LLC Figure 9.15. They are inactive by themselves and are acti- p21 vatedNOT by the FOR binding SALE of cell OR cycle–specific DISTRIBUTION proteins called NOT p27FOR SALE OR DISTRIBUTION cyclins. This means that the CDKs can be synthesized in advance and left in the cytoplasm. In addition to cyclins, the Cyclin CDK CDKs are regulated by multiple phosphorylation events. One Active © Jones &set Bartlettof kinases, theLearning, Wee1 family LLC of ser/thr kinases, inhibits the© Jones & Bartlett Learning, LLC CDKs, while another, the CDK-activating kinases (CAKs), Figure 9.15 Formation of an active CDK requires binding to a NOT FORactivates SALE them. OR (Wee1DISTRIBUTION kinases inhibit cell cycle progression,NOT cyclin.FOR The SALE process OR is regulated DISTRIBUTION by positive and negative factors.

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DNA Damage from degradation. This leads to an increased level of p53 UV X ray and activates its ability to serve as a transcription factor to © Jones & Bartlett Learning, LLC © Jonesturn on& someBartlett genes Learning, and repress other LLC genes. Among those NOT FOR SALE OR DISTRIBUTIONATRAϩ TM Chk 1ϩ2 NOT genesFOR turned SALE on ORare GADD45 DISTRIBUTION to stimulate DNA repair; p21/ WAF-1, whose product binds to and inhibits the CDK/cyclin complexes for G1 arrest (or promotes apoptosis if the DNA Cdc 25 damage is too great); sets of large intergenic noncoding RNAs GF p53 (lincRNAs) to mediate transcription repression; and miRNAs © Jones & Bartlett Learning, LLC(as described in the chapter titled© Jones Regulatory & RNA Bartlett). A specific Learning, LLC NOT FOR SALE OR DISTRIBUTIONlincRNA, p21-lincRNA, mediatesNOT the FOR repressive SALE properties OR DISTRIBUTIONof GADD45 myc p21 MDM2 p19 p53 by binding to specific chromatin complexes. repair DNA damage also independently activates a pair of protein kinases, Chk1 and Chk2, which phosphorylate and inhibit Rb CDKs, and phosphorylate and inhibit the phosphatase Cdc25 © Jones & Bartlett Learning, LLC (cell division© cycle), Jones which & isBartlett required to Learning, activate the CDKs. LLC Figure 9.16 DNA damage pathway. p53 is activated by DNA damage.NOT Activated FOR p53 SALE halts the OR cell cycleDISTRIBUTION through Rb and NOT FOR SALE OR DISTRIBUTION stimulates DNA repair. p53 is regulated by a complex set of 9.11 Checkpoint Control activators and inhibitors. for Entry into S Phase: Rb, a the cell size and absence of DNA damage criteria are met. Even Guardian of the Checkpoint © Jones &in theBartlett presence Learning, of an oncogenic LLC mutant RAS protein, tumor© Jones & Bartlett Learning, LLC NOT FORsuppressors SALE OR will DISTRIBUTIONprevent the cell from progressing from G1 toNOT FOR SALE OR DISTRIBUTION S; they are the brakes on the cell cycle. Mutations in tumor Key Concepts suppressor proteins allow damaged and undersized cells to replicate. These recessive, loss-of-function mutations, especially • Rb is the major guardian of the cell cycle, integrating information about DNA damage and cell growth. in p53 and Rb, are the most common tumor suppressor muta- © Jones & Bartlett Learning, LLC• Rb binds the activation domains© Jones of a set of& essential Bartlett Learning, LLC tions in tumors; frequently both are seen together. transcription factors, the E2F family, in the cytoplasm to The DNA damage NOTcheckpoint FOR controlled SALE ORby p53 DISTRIBUTION is the prevent them from turning onNOT the genes FOR required SALE for ORcell DISTRIBUTION one that is best understood (Figure 9.16). The function of cycle progression. p53 is to relay information to the CDK/cyclins that damage • When Rb is phosphorylated by a cyclin/CDK complex, it has occurred to prevent entry into S phase; that is, it ulti- releases E2F to permit cell cycle progression. mately causes cell cycle arrest. In addition, in the event that damage© Jonesis very extensive & Bartlett or otherwise Learning, unrepairable, LLC p53 will Let’s now ©examine Jones how & Bartlettan undamaged Learning, cell progresses LLC initiateNOT an alternate FOR pathway,SALE ORapoptosis DISTRIBUTION, or programmed cell through G1NOT (Figu rFORe 9.17 ).SALE A growth OR factor DISTRIBUTION signal, executed death (PCD). p53 transcription is upregulated by growth through the signal transduction pathway, is required to turn factor stimulation, as the cell begins preparation for its trip on the gene for the first cyclin expressed, Cyclin D (humans through G1 and the important G1 to S transition. have three different forms of this gene while Drosophila The p53 protein product is regulated by multiple com- has one). Its partners, already in the cytoplasm, are CDK4 © Jones &plex Bartlett pathways. Learning,The major regulator LLC is a protein called MDM2,© Jonesand -6. & CyclinsBartlett are theLearning, positive regulators LLC of the CDK pro- NOT FORwhich SALE works OR through DISTRIBUTION a negative feedback loop. MDM2 tran-NOT teinFOR kinases; SALE by ORthemselves DISTRIBUTION CDKs are inactive. Cyclin D is scription is increased by p53, and it in turn inhibits p53 in a pos- required for entry into S phase. Growth factor must be con- itive feedback loop, by targeting it to the ubiquitin-dependent tinuously present for at least the first half of G1. proteosomal degradation pathway, as described further in the The key for cell cycle progression is the tumor suppressor section Checkpoint Control for Entry into S Phase: Rb, a Guard- protein Rb. Although Rb has multiple roles in the nucleus as ian of the Checkpoint coming© Jones up next. & It Bartlettalso binds toLearning, p53 and LLCdirect regulator of chromatin structure© Jones and & transcription, Bartlett Learning, we LLC prevents it from activatingNOT transcription. FOR SALE DNA ORdamage DISTRIBUTION leads focus in this section on its roleNOT in the FOR cytoplasm SALE as the OR major DISTRIBUTION to phosphorylation of MDM2, which inhibits its ability to pro- guardian of entry into S phase. Rb binds to the transcrip- mote p53 degradation, allowing p53 levels to increase. Growth tion factor E2F and inhibits its ability to enter the nucleus to factor stimulation of cell cycle progression also leads to an turn on those genes required for progression through G1 and increase in transcription of the p19ARF protein (p14 in humans), entry into S phase. Within G1 is a critical point controlled by which© binds Jones to and & Bartlettinhibits MDM2’s Learning, ability toLLC inhibit p53. Rb, called the© Jonesrestriction & point Bartlett or START Learning, point (different LLC in The humanNOT FORp14ARF isSALE transcribed OR fromDISTRIBUTION an interesting genetic different species),NOT FORat which SALE the cell OR becomes DISTRIBUTION committed to locus, the INK4a/ARF locus, which gives rise to three proteins continuing through the cell cycle. Ultimately, Rb integrates by alternative splicing and alternative promoter usage: p15INK, signals concerning both DNA damage as described in the p16INK, and p14ARF (ARF stands for alternate reading frame). section on p53, and cell size (or growth of the cell) pathways p53 is activated by DNA damage or different kinds and is thus the key guardian of progression to S phase. © Jones &of Bartlettstress through Learning, a protein LLCkinase relay system from the© JonesFor & cellBartlett cycle progression Learning, to occur, LLC Rb must be phospho- NOT FORnucleus SALE that OR ultimately DISTRIBUTION phosphorylates and stabilizes p53NOT rylatedFOR SALEby CDK/cyclin; OR DISTRIBUTION phosphorylation of Rb releases E2F.

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Growth Factor phase. Among the genes activated is the E2F gene to increase the abundance of the E2F protein and Cyclin E. Cyclin E is © Jones & Bartlett Learning, LLC © Jonesactivated & Bartlett by the middle Learning, of G1, and LLCit is also required for pro- NOT FOR SALE OR DISTRIBUTIONRAS RAF NOT gressionFOR SALE into S phase,OR DISTRIBUTION adding to and amplifying the initial phosphorylation of Rb. Finally, just before S phase begins, Cyclin A is synthesized, and it is also required for entry and MYC, JUN/FOS continuation through S phase. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Cyclin Dϩ CDK NOT FOR SALE OR DISTRIBUTIONSummary NOT FOR SALE OR DISTRIBUTION p21 p53 Chk 1ϩ2 Cdc 25 • A fixed time of 40 minutes is required to replicate p27 : size control the E. coli chromosome, and an additional 20 minutes is required before the cell can divide. When © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Rb•E2F cells divide more rapidly than every 60 minutes, a NOT FOR SALE OR DISTRIBUTIONE2F replicationNOT FOR cycle SALEis initiated OR before DISTRIBUTION the end of the Rb•P preceding division cycle. This generates multiforked chromosomes. The initiation event occurs Progression once and at a specific time in each cell cycle. Initia- through G1 tion timing depends on accumulating the active ini- © Jones & Bartlett Learning, LLC © Jones & Bartletttiator protein Learning, DnaA and onLLC inhibitors that turn off NOT FOR SALE OR DISTRIBUTION NOT FOR SALEnewly synthesized OR DISTRIBUTION origins until the next cell cycle. Cyclin E • E. coli grows as a rod-shaped cell that divides into daughter cells by formation of a septum that forms at mid-cell. The shape is maintained by an envelope Entry into S of peptidoglycan that surrounds the cell. The rod © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Figure 9.17 Growth factors are required to start the cell shape is dependent on the MreB actin-like protein cycle and continue into S NOTphase. TheFOR CDK-cyclin SALE complex OR DISTRIBUTION that forms a scaffold NOTfor recruiting FOR theSALE enzymes OR nec DISTRIBUTION- phosphorylates Rb to cause it to release the transcription factor essary for peptidoglycan synthesis. The septum is E2F to go into the nucleus to turn on genes for progression dependent on FtsZ, which is a tubulin-like protein through G1 and into S phase. that can polymerize into a filamentous structure called a Z-ring. FtsZ recruits the enzymes necessary The ultimate© Jones control & ofBartlett cell cycle progressionLearning, is thus LLC the regula- to ©make Jones the septum. & Bartlett Absence Learning, of septum formation LLC tion ofNOT CDK activityFOR SALE by a set ofOR inhibitor DISTRIBUTION proteins, CKIs (cyclin generatesNOT FOR multinucleated SALE OR filaments; DISTRIBUTION an excess of kinase inhibitors). p21, induced by DNA damage through septum formation generates anucleate minicells. p53, is a CKI. It is the major link between the DNA damage • Many transmembrane proteins interact to form the checkpoint and Rb. Another major CKI is p27, a member septum. ZipA is located in the inner bacterial mem- of the Cip/Kip family. It is present in fairly high levels in G0 brane and binds to FtsZ. Several other fts products, © Jones &cells Bartlett to prevent Learning, activation to G1.LLC EGFR activation leads to its© Jones & Bartlettmost of which Learning, are transmembrane LLC proteins, join the NOT FORreduction. SALE ORp27 is DISTRIBUTION also activated in G1 by the cytokine TGF-3,NOT FOR SALEZ-ring inOR an DISTRIBUTIONordered process that generates a septal a major growth inhibitor. p19/p16/INK/ARF is another ring. The last proteins to bind are the SEDS protein major class of CKI proteins that control Cyclin D activity FtsW and the transpeptidase FtsI (PBP3), which (these two different proteins, INK and ARF, are made from together function to produce the peptidoglycans the same gene from alternate reading frames). of the septum. Chromosome segregation involves Cell size or growth ©of theJones cell is &monitored Bartlett by aLearning, titration LLC several processes, including© Jones separation & Bartlett of catenated Learning, LLC mechanism. A cell enteringNOT G1 FOR has aSALE fixed set OR of differentDISTRIBUTION products by topoisomerases,NOT FOR site-specific SALE recombi OR DISTRIBUTION- classes of CKI proteins to prevent cell cycle progression. For nation, and the action of MukB/SMC proteins in the cell to progress through G1, this inhibition must be over- chromosome condensation following DNA replica- come by the synthesis of more Cyclin D. The length of G1 is tion. Plasmids and bacteria have site-specific recom- determined by how long it takes to synthesize a sufficient level bination systems that regenerate pairs of monomers of cyclins© Jones to overcome & Bartlett the level of Learning, CKIs. LLC by© resolving Jones dimers& Bartlett created Learning, by general recombi LLC - DuringNOT FORG1, three SALE different OR cyclins DISTRIBUTION are made. Cyclin D, as nation.NOT The FOR Xer SALE system actsOR onDISTRIBUTION a target sequence described earlier, is the first synthesized, activated by growth located in the terminus region of the chromosome. factor. As the cell continues to grow, the level of Cyclin D The system is active only in the presence of the FtsK reaches a point of titrating out the CKIs, and the Cyclin D/ protein of the septum, which might ensure that it cdk4/6 complex can begin phosphorylating Rb/E2F. This acts only when a dimer needs to be resolved. © Jones &will Bartlett cause Rb toLearning, begin to release LLC E2F, which can then activate© Jones &• BartlettThe eukaryotic Learning, cell cycle LLCis governed by a complex NOT FORgenes SALE for progression OR DISTRIBUTION through the cell cycle and ultimately SNOT FOR SALEset of regulatory OR DISTRIBUTION factors. Licensing to begin the cell

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cycle, as opposed to enter or remain in G0, requires Reyes-Larnothe, R., et al. (2008). Escherichia coli and its chromo- a positive growth factor signal interacting with its some. Trends Microbiol. 16, 238–245. © Jones & Bartlettreceptor Learning, to initiate the LLC signal transduction pathway.© JonesResearch & Bartlett Learning, LLC NOT FOR SALEThis OR biochemical DISTRIBUTION relay of information from outsideNOT Dominguez-Escobar,FOR SALE OR J., DISTRIBUTION et al. (2011). Processive movement of the cell through the RAS-GTP and RAF protein MreB-associated cell wall bio-synthetic complexes in bacteria. kinase ultimately results in the activation of a set Science, 333, 225–228. of transcription factors in the cytoplasm. These can Garner, E. C., et al. (2011). Coupled, circumferential motions of the then enter the nucleus to begin the transcription of cell wall synthesis machinery and MreB filaments in B. subtilis. Science 333, 222–225. genes required© for Jones the progression & Bartlett through Learning, G1 and LLC © Jones & Bartlett Learning, LLC Spratt, B. G. (1975). Distinct penicillin binding proteins involved in ultimate entryNOT into S FORphase SALEand replication OR DISTRIBUTION of the the division, elongation, andNOT shape FOR of E. coli SALE K12. Proc. OR Natl. DISTRIBUTION chromosomes. Acad. Sci. USA 72, 2999–3003. • The cell cycle—that is, progression from G1 to S phase and beyond—is regulated primarily by phos- 9.4 Mutations in Division or Segregation Affect phorylation events carried out by a set of protein Cell Shape © Joneskinases, the& BartlettCDKs, and Learning, balanced by phosphatases.LLC © Jones & Bartlett Learning, LLC Research NOTThe FOR kinases SALE are controlled OR DISTRIBUTION by a set of cell cycle NOT FOR SALE OR DISTRIBUTION stage–specific proteins called cyclins that bind to Adler, H. I., et al. (1967). Miniature E. coli cells deficient in DNA. Proc. Natl. Acad. Sci. USA 57, 321–326. the CDKs and convert an inactive CDK into an Niki, H., et al. (1991). The new gene mukB codes for a 177 kd pro- active kinase. Progression through G1 into S phase tein with coiled-coil domains involved in chromosome parti- is allowed only if there is no DNA damage and the tioning of E. coli. EMBO J. 10, 183–193. © Jones & Bartlettcell has Learning, grown a sufficient LLC amount in size. These© Jones & Bartlett Learning, LLC two requirements are enforced by a pair of tumor- NOT FOR SALE OR DISTRIBUTION NOT 9.5FOR FtsZ SALE Is Necessary OR DISTRIBUTION for Septum Formation suppressor proteins. p53 guards the DNA damage Reviews checkpoint to prevent the replication of damaged DNA. Rb is the guardian that integrates DNA dam- Errington, J., et al. (2003). Cytokinesis in bacteria. Microbiol Mol. Biol. Rev. 67, 52–65. age and cell-size information to ultimately control © Jones & Bartlett Learning, LLCWeiss, D. S. (2004). Bacterial cell© division Jones and &the Bartlett septal ring. Mol.Learning, LLC whether the gene regulator E2F is allowed into the Microbiol 54, 588–597. nucleus to beginNOT transcription. FOR SALE OR DISTRIBUTIONResearch NOT FOR SALE OR DISTRIBUTION Bi, E. F., and Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. Nature 354, 161–164. References Mercer, K. L., and Weiss, D. S. (2002). The E. coli cell division protein FtsW is required to recruit its cognate transpeptidase, FtsI 9.2 Bacterial© Jones Replication & Bartlett I sLearning, Connected LLC to the (PBP3),© to Jonesthe division & site. Bartlett J. Bacteriol Learning, 184, 904–912. LLC Cell NOTCycle FOR SALE OR DISTRIBUTION Pichoff, S., andNOT Lutkenhaus, FOR J.SALE (2002). UniqueOR DISTRIBUTION and overlapping roles Reviews for ZipA and FtsA in septal ring assembly in Escherichia coli. Haeusser, D. P., and Levin, P. A. (2008). The great divide: coordinat- EMBO J. 21, 685–693. ing cell cycle events during bacteria growth and division. Curr. Opin. Microbiol. 11, 94–99. 9.6 min and noc/slm Genes Regulate the © Jones &Scalfani, Bartlett R. A., andLearning, Holzen, T. M.LLC (2007). Cell cycle regulation of© JonesLocation & Bartlett of the S Learning,eptum LLC DNA replication. Annu. Rev. Gen. 41, 237–280. NOT FORResearch SALE OR DISTRIBUTION NOT RFOReviews SALE OR DISTRIBUTION Adams, D. W., et al. (2014). Cell cycle regulation by the bacterial Donachie, W. D., and Begg, K. J. (1970). Growth of the bacterial cell. nucleoid. Curr. Opin. Microbiol. 22, 94–101. Nature 227, 1220–1224. Lutkenhaus, J. (2007). Assembly dynamics of the bacterial MinCDE Lobner-Olesen, et al. (1989). The DnaA protein determines the ini- system and spatial regulation of the Z Ring. Annu. Rev. tiation mass of Escherichia coli. K-12. Cell 57, 881–889. © Jones & Bartlett Learning, LLCBiochem. 76, 539–562. © Jones & Bartlett Learning, LLC Research 9.3 The Shape and SNOTpatial FOR Organization SALE OR of aDISTRIBUTION NOT FOR SALE OR DISTRIBUTION Bernhardt, T. G., and de Boer, P. A. J. (2005). SlmA, a Bacterium Are Important during Chromosome nucleoid-associated, FtsZ binding protein required for Segregation and Cell Division blocking septal ring assembly over chromosomes in E. coli. Reviews Mol. Cell 18, 555–564. Eraso,© J. M.,Jones and Margolin, & Bartlett W. (2011). Learning, Bacterial cell LLC wall: thinking Fu, X. L., et al.© (2001). Jones The &MinE Bartlett ring required Learning, for proper placement LLC globally, acting locally. Curr. Biol. 21, R628–R630. of the division site is a mobile structure that changes its cellular Eun, Y.-J.,NOT et al.FOR (2015). SALE Bacterial OR filament DISTRIBUTION systems: toward under- locationNOT during FOR the Escherichia SALE coliOR division DISTRIBUTION cycle. Proc. Natl. standing their emergent behavior and cellular functions. Acad. Sci. USA 98, 980–985. J. Biol. Chem. 290, 17181–17189. Raskin, D. M., and de Boer, P. A. J. (1999). Rapid pole-to-pole oscil- Osborn, M. J., and Rothfield, L. (2007). Cell shape determination in lation of a protein required for directing division to the middle Escherichia coli. Curr. Opin. Microbiol. 10, 606–610. of Escherichia coli. Proc. Natl. Acad. Sci. USA 96, 4971–4976. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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9.7 Partition Involves Separation of the Nan, X., et al. (2015). Ras-GTP Dimers Activate the Mitogen- Chromosomes Activated Protein Kinase (MAPK) Pathway. Proc. Natl. Acad. Sci. USA 112, 7996–8001. © Jones Reviews& Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Zhou Y., et al. (2015). Membrane potential modulates plasma mem- NOT FORBouet, SALE J. Y., etOR al. (2014). DISTRIBUTION Mechanisms for chromosome segregation.NOT FORbrane SALE phospholipid OR DISTRIBUTION dynamics and K-Ras signaling. Science Curr. Opin. Microbiol. 22, 60-65. 349, 873–876. Draper, G. C., and Gober, J. W. (2002). Bacterial chromosome segregation. Annu. Rev. Microbiol. 56, 567–597. 9.10 Checkpoint Control for Entry Into S Phase: Research p53, a Guardian of the Checkpoint Case, R. B., et al. (2004). ©The Jones bacterial &condensin Bartlett MukBEF Learning, com- LLC © Jones & Bartlett Learning, LLC pacts DNA into a repetitive,NOT FOR stable SALE structure. OR Science DISTRIBUTION 305, Reviews NOT FOR SALE OR DISTRIBUTION 222–227. Kruse, J. P., and Gu, W. (2009). Modes of p53 regulation. Cell 1367, Danilova, O., et al. (2007). MukB colocalizes with the oriC region 609–622. and is required for organization of the two Escherichia coli Scott, J. D., and Pawson, T. (2009). Cell signaling in space and time: chromosome arms into separate cell halves. Mol. Microbiol. where proteins cometogether and when they’re apart. Science 65,© 1485–1492.Jones & Bartlett Learning, LLC 326, 1220–1224.© Jones & Bartlett Learning, LLC Fisher, J. K., et al. (2013). Four-dimensional imaging of E. coli Vousden, K. H. (2000). p53 death star. Cell 103, 691–694. nucleoidNOT FORorganization SALE and ORdynamics DISTRIBUTION in living cells. Cell 153, ResearchNOT FOR SALE OR DISTRIBUTION 882–895. Agami, R., and Bernards, R. (2000). Distinct initiation and mainte- Jacob, F., et al. (1966). On the association between DNA and the nance mechanisms cooperate to induce G1 cell cycle arrest in membrane in bacteria. Proc. Roy. Soc. Lond. B. Bio. Sci. 164, response to DNA damage. Cell 102, 55–66. 267–348. Hemann, M. T., et al. (2005). Evasion of the p53 tumor surveil- © Jones &Sawitzke, Bartlett J. A., andLearning, Austin, S. (2000). LLC Suppression of chromosome© Joneslance & Bartlett network by Learning, tumor-derived LLCMYC mutants. Nature 436, NOT FOR SALEsegregation OR defectsDISTRIBUTION of E. coli muk mutants by mutations inNOT FOR807–812. SALE OR DISTRIBUTION topoisomerase I. Proc. Natl. Acad. Sci. USA 97, 1671–1676. Huarte, M., et al. (2010). A large intergenic noncoding RNA Wang, X., et al. (2014). Bacillus subtilis chromosome organization induced by p53 mediates global gene repression in the p53 oscillates between two distinct patterns. Proc. Natl. Acad. Sci. response. Cell 142, 409–419. USA 111, 12877–12882. Jin, L., et al. (2011). micoRNA-149*, a p53-responsive microRNA, © Jones & Bartlett Learning, LLCfunctions as an oncogenic© regulator Jones in &human Bartlett melanoma. Learning, LLC 9.8 Chromosomal Segregation May Require Proc. Natl. Acad. Sci. USA 108, 15840–15845. Site-Specific ecombinationR NOT FOR SALE OR DISTRIBUTIONPurvis, J. E., et al. (2012). P53 dynamicsNOT FOR control SALE cell fate. OR Science DISTRIBUTION Research 336, 1440–1444. INK4a Aussel, L., et al. (2002). FtsK is a DNA motor protein that activates Sun, P., et al. (2010). GRIM-19 and p16 synergistically regulate chromosome dimer resolution by switching the catalytic state cell cycle progression and E2F1-responsive gene expression. J. Biol. Chem. 285, 27545–27552. of© the Jones XerC and & XerD Bartlett recombinases. Learning, Cell 108, 195–205.LLC © Jones & Bartlett Learning, LLC Stouf, M., et al. (2013). FtsK actively segregates sister chromosomes Sun, L., et al. (2009). JFK, a Kelch domain-containing F-box pro- inNOT Escherichia FOR coli SALE. Proc. Natl. OR Acad. DISTRIBUTION Sci. USA 110, 11157–11162. tein, linksNOT the SCF FOR pathway SALE to p53 OR regulation. DISTRIBUTION Proc. Natl. Acad. Sci. USA 106, 10195–10200. Weber J. D., et al. (2000). p53-Independent functions of the p19ARF 9.9 The Eukaryotic Growth Factor Signal tumor suppressor. Genes Dev. 14, 2358–2365. Transduction Pathway Promotes Entry to S Phase © Jones & Bartlett Learning, LLC © Jones9.11 Checkpoint& Bartlett Learning,Control for ELLCntry Into S Phase: Reviews Rb, a Guardian of the Checkpoint NOT FORGood, SALE M. C., OR et al.DISTRIBUTION (2011). Scaffold proteins: hubs for controllingNOT FOR SALE OR DISTRIBUTION the flow of cellular information. Science 332, 680–686. Reviews Kyriakis, J. M. (2009). Thinking outside the box about Ras. J. Biol. Enders, G. H. (2008). Expanded roles for chk1 in genome mainte- Chem. 284, 10993–10994. nance. J. Biol. Chem. 283, 17749–17752. Kip1 Oda, K., et al. (2005). A comprehensive pathway map of epidermal Kaldis, P. (2007). Another piece of the p27 puzzle. Cell 128, growth factor receptor© signaling. Jones Mol. & Syst.Bartlett Biol. 1, Epub.Learning, LLC241–244. © Jones & Bartlett Learning, LLC Research Weinberg, R. A. (1995). The Retinoblastoma protein and cell cycle NOT FOR SALE OR DISTRIBUTIONcontrol. Cell 81, 323–330. NOT FOR SALE OR DISTRIBUTION Alvarado, D., et al. (2010). Structural basis for negative cooperativity in growth factor binding to an EGF receptor. Cell 142, Research 568–579. Deng, C., et al. (1995). Mice lacking p21CIP1/WAF1 undergo nor- Coskun, Ü., et al. (2011). Regulation of human EGF receptor by lip- mal development, but are defective in G1 checkpoint control. ids.© JonesProc. Natl. &Acad. Bartlett Sci. USA 108,Learning, 9044–9048. LLC Cell 82,© 675–684. Jones & Bartlett Learning, LLC Gao, Y. S., et al. (2010). The Microtubule-associated Histone Janbandhu, V. C., et al. (2010). p65 negatively regulates transcrip- DeacetylaseNOT FOR 6 (HDAC6) SALE ORregulates DISTRIBUTION epidermal growth factor tion of NOTthe Cyclin FOR E gene. SALE J. Biol. Chem.OR DISTRIBUTION 285, 17453–17464. WAF1/CIP1 receptor (EGFR) endocytic trafficking and degradation. J. Biol. Kan, Q., et al. (2008). Cdc6 determines utilization of p21 - Chem. 285, 11219–11226. dependent damage checkpoint in S phase cells. J. Biol. Chem. Misaki, R., et al. (2010). Palmitoylation directs Ras proteins to the 283, 17864–17872. correct intracellular organelles for trafficking and activity. Koepp, D. M., et al. (2001). Phosphorylation-dependent ubiquitination Fbw7 © Jones & BartlettJ. Cell Biol. Learning,191, 23–29. LLC © Jonesof & Cyclin Bartlett E by the SCFLearning, ubiquitin LLC ligase. Science 294, 173–177. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

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