Chromosome Structure Introductory article

N Patrick Higgins, University of Alabama, Birmingham, Alabama, USA Article Contents

. Abundance Genes are organized into discrete cellular structures called chromosomes that coordinate . Chromosome Size DNA replication and distribution of replicated genetic copies between two daughter cells. . Plasmids As vehicles of genetic transmission, chromosomes play a central role in Darwinian . Chromosome Shape evolution. . Enzymes of DNA Topology . Chromatin Abundance . DNA Replication . Eukaryotic Segregation Biology is divided into three great kingdoms: Eubacteria, . Transcription Archaea and Eukaryota. Bacteria (Eubacteria and Ar- . Recombination chaea) are ubiquitous in the environment, and these small . Chi Sites single-celled organisms grow over an amazingly wide range . Transposons ofenvironmental conditions. For example, bacteria can . Site-specific Recombination grow at temperatures below freezing, and certain members ofthe archaea can grow at depths ofover three miles and at temperatures exceeding 6008F and 200 atmospheres of pressure. Eukaryotes, which include the more conspicuous genome yet analysed is that of Myxococcus xanthus, with fungi and all plants and animals, are usually recognized as 9.2 Mbp (9 200 000 bp). However, the best studied organ- the predominant life forms on earth. They are subjects of ism in nature is E. coli, which has a 4.6-Mbp chromosome great biological interest and they differ from bacteria by with 4288 genes for proteins, seven operons for ribosomal having a larger cell size and by compartmentalizing ribonucleic acids (RNAs), and 86 genes for transfer RNAs. chromosomal deoxyribonucleic acid (DNA) in a nucleus, The E. coli chromosome contains numerous gene which separates it from the protein-making cytoplasm. families, each family having evolved from a common None the less, considering the large cell numbers and wide ancestor. The largest gene family is comprised of 96 three- environmental growth ranges, bacteria easily account for component (ABC) transporters, which are membrane- the majority ofDNA biomass on earth. bound machines that import and export a variety ofsmall The chromosome is the heart ofa central paradox in molecules and proteins. By contrast, the smallest free- evolution. How do species in the three kingdoms remain living organism is Mycoplasma genatalium with a 0.58- the same over long periods ofgeological time and also Mbp genome that encodes 468 protein genes, one generate sufficient variability to produce new species, ribosomal RNA operon and one ABC transporter. Both sometimes relatively rapidly? Stability versus change is a E. coli and M. genatalium have complete information for crucial dichotomy in molecular biology. The events that the synthesis ofcell walls, cell membranes and critical bring about stability and change in DNA structure involve enzymes ofintermediary metabolism, plus the RNA processes ofreplication, transcription and recombination. molecules, ribosomal proteins and a clutch ofenzymes Similar mechanisms operate in the three living kingdoms, (the replisome) to replicate DNA efficiently. Putative but the key molecular mechanisms that control and functions for about a quarter of the genes of E. coli remain catalyse these events are understood best in a eubacterium: to be discovered. A reasonable estimate is that 150–200 Escherichia coli. protein-encoding genes would be ‘essential’ for a basic bacterial lifestyle (in a rich medium). Eukaryotic organisms generally have larger chromo- somes than bacteria. For example, the yeast Saccharo- myces cerevisiae has about 6000 genes (50% more than E. Chromosome Size coli), whereas mammalian cells contain 1000 times (per haploid equivalent) the DNA ofan E. coli cell. In humans Free-living bacteria need genetic information to synthesize the 5000 Mbp ofhaploid DNA is distributed among 22 proteins for executing vital functions. Most bacteria have a autosomes and two sex-specific chromosomes. Eukaryotic single chromosome with DNA that is about 2 Mbp (mega DNA is localized in a compartment, the nucleus, which is base pairs) long (1 Mbp 5 1 000 000 base pairs), but the separated by a phospholipid-containing membrane from DNA content ofdifferent species varies from0.58 to cytoplasmic ribosomes and protein translation activity. greater than 9 Mbp ofDNA, and some bacteria have During cell division, the eukaryotic nuclear membrane multiple chromosomes. For example, Leptospira has two chromosomes of4.4 and 4.6 Mbp and the largest bacterial

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 1 Chromosome Structure

breaks down once per cell cycle to distribute the 46 diploid Plectonemic Paranemic chromosomes equally between two daughter cells.

Plasmids 5kbp In addition to the large chromosome, many (most?) Plasmid bacteria have additional DNA molecules called plasmids (or episomes.) Plasmids are separate DNA molecules that contain a replication origin which allows them to multiply independently ofthe host chromosome. Plasmids range in size from 1 kbp (Kilo base pair) (1000 bp) to 100 kbp, and these DNA molecules encode genetic systems for specia- Eukaryotic chromatin Prokaryotic chromatin lized functions. Some plasmids make extracellular appen- Gyrase dages that allow bacteria to infect and colonize sensitive Histone eukaryotic hosts. Plasmids often carry genes that confer on Solenoidal assembly bacteria the ability to survive in the presence ofantibiotics supercoils σ such as tetracycline, kanamycin and penicillin. Many = –0.05 HU H-NS plasmids also contain genes that promote DNA transfer so Deproteinate that plasmid genes can move into other bacterial species. Plasmid transfer has caused the emergence of bacterial pathogens that are resistant to most ofthe useful Interwound antibiotics in medicine, with notable examples including supercoils (a) σ = –0.05 multidrug-resistant strains of Staphylococcus and Myco- bacterium tuberculosis. In most eukaryotes, plasmids are rare. However, S. Topo cerevisiae contains a plasmid called the 2-m circle which IV efficiently partitions to new daughter cells at every cell Topo division. This DNA serves as a convenient module for gene II cloning and performing genetic experiments in yeast. (b) Figure 1 (a) DNA is a plectonemic helix (left) rather than a paranemic helix (right), and thus it must spin axially to undergo replication, transcription and extended pairing for homologous recombination. Chromosome Shape Supercoiling in circular bacterial chromosomes is maintained by the concerted action of DNA gyrase, which introduces negative supercoils at On a macroscopic scale, bacterial chromosomes are either the expense of adenosine triphosphate (ATP) binding and hydrolysis, and circular or linear. Circular chromosomes are most TopoI plus TopoII, which remove excess negative supercoils. Negative common, at least among the best-studied bacteria. How- supercoiled DNA adopts an interwound conformation. However, ‘solenoidal’ supercoils can be stabilized when DNA is wrapped on a protein ever, the causative agent ofLyme disease, Borrelia surface. This is the mechanism of supercoil formation in mammalian cells burgdorphei, has a 2-Mb linear chromosome plus 12 (centre left). Most bacteria have nonspecific DNA-binding proteins such as different linear plasmids. Eukaryotic chromosomes are HU and H-NS, which stabilize supercoils but are much less effective at invariably linear, and they have two ends, each carrying a condensing DNA compared with true histones (centre right). (b) Bacteria special structure called a telomere, and a organized region and eukaryotes both have an enzyme, TopoIV and TopoII respectively, designed to unknot and untangle DNA. called the centromere which allows the chromosome to attach to cellular machinery that moves it to the proper place during cell division. DNA synthesis the rotation speed approaches 6000 rpm. One critical facet of chromosome structure is that DNA Chromosomal DNA molecules are very long and thin, so is a plectonemic helix, which means that two helical strands DNA must fold many times to fit within the confines of a entwine about each other. For duplex DNA the two bacterial cell. How does DNA twisting transpire without antiparallel strands, often referred to as the Watson and chromosomes getting tangled up? The problem was Crick (red and blue in Figure 1), are interwound once for important enough that Watson and Crick considered a every 10 bp. Because ofthis wound configuration, bio- paranemic helix, which is a pair ofcoils that lay side by side chemical transactions that involve strand separation without interwinding. Strands ofa paranemic helix can require chromosome movement (spin) about DNA’s long separate without rotation (Figure 1). None the less DNA is axis. The processes ofDNA replication, recombination plectonemic, and keeping chromosomes untangled re- and transcription all require DNA rotation, and during quires a special class ofenzymes called topoisomerases.

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net Chromosome Structure

Enzymes of DNA Topology enzyme such as gyrase, eukaryotic DNA is wrapped tightly around nucleosomes, generating solenoidal supercoils that The enzymes that solve the untangling problem are condense DNA 8-fold (Figure 1). A nucleosome contains topoisomerases. Topoisomerases break and rejoin DNA four subunits: histones H2A, H2B, H3 and H4. If histones molecules, thereby allowing individual strands to pass one are stripped from DNA by protein denaturants, eukaryotic through another. The number ofphosphodiester bonds DNA adopts the interwound structure ofbacterial broken and reformed per reaction cycle divides topoi- chromosomes, with about the same negative superhelix somerases into two classes. Type I enzymes, which include density (Figure 1). TopoI and TopoIII (the odd-numbered topoisomerases), Eukaryotes have three topoisomerases: TopoI and break one strand per cycle, and type II enzymes (even TopoIII are type 1 enzymes that break only one strand, numbered), gyrase, eukaryotic TopoII, and E. coli and TopoII is the only type 2 activity that breaks both TopoIV, break two strands simultaneously. These en- strands simultaneously. Eukaryotic TopoI removes both zymes have two important roles: they provide a swivel to positive and negative supercoils from DNA, whereas allow processes such as replication and transcription to TopoII carries out unknotting and decatenating reactions proceed unimpeded, and they untangle knots and inter- (see below). TopoI and TopoII are essential for eukaryotic chromosome links between DNA molecules. cell viability; the functions of TopoIII in both eukaryotes In all organisms DNA becomes organized into super- and bacteria remain undefined. coils, which are turns ofthe double strand over the interwound twists ofthe Watson–Crick helix. Supercoils can be either positive or negative; negative supercoils are opposite to the handedness ofthe Watson–Crick turns, Chromatin and positive supercoils have the same handedness. Super- coil density is defined by a term s, which represents the Chromatin is a term that refers not just to DNA but to the number ofsuperhelical turns divided by the Watson–Crick proteins attached to a chromosome. In the dimensions of turns ofa double helix. Supercoiling influences the B-form DNA, E. coli is a sphere that is 6 kbp long (8 nm) Watson–Crick structure and, like the spring, the free and 4 kbp wide (2 nm), so the 4.6-Mbp chromosome must energy ofsupernegative supercoils increases exponentially be folded many times to fit within a cell. Negative with quantity. Enzymes that unwind DNA to carry out supercoiling forces DNA into an interwound configura- their function (RNA polymerase and DNA replisomes) tion (Figure 1). Interwound supercoiling is produced at the may sense and utilize supercoiling energy. However, once expense ofATP by the enzyme gyrase, and supercoiling negative supercoiling has reached a critical density, the produces two important consequences. First, the DNA Watson–Crick structure will unwind locally, forcing DNA molecule doubles back on itselfso that length is halved into alternative structures to relieve tension. Well-studied relative to that ofthe linear form.Second, supercoiled supercoil-dependent alternatives to the Watson–Crick branches are dynamic so that opposing DNA sites in the form of DNA include left-handed Z-DNA, cruciforms, interwound network are constantly changing. A protein and triple-stranded or H-form DNA. bound to DNA in one supercoiling domain interacts more In eubacteria, gyrase, TopoI and TopoIV maintain in than 100 times more frequently with other proteins in the vivo supercoiling levels, s 520.05 to 2 0.075. Gyrase is same domain than it does with proteins bound to a unique for its ability to introduce negative supercoils into different domain. When DNA is liberated from cells by relaxed, positively or negatively supercoiled DNA at the breaking the peptidoglycan coat, chromosomes form expense ofadenosine triphosphate (ATP) binding and bundled loops that represent domains. Such preparations hydrolysis. An essential enzyme in bacteria, gyrase is (called nucleoids) behave as discrete bodies. critical for DNA reactions that include recombination, Many reactions ofthe chromosome require the forma- replication, transcription and chromosome segregation. In tion ofintricate DNA–protein machines to replicate, vitro, DNA gyrase can supercoil DNA to a value of transcribe or recombine DNA at specific sequences. A s 520.1, a level at which many sequences adopt an group of‘chromosome-associated’ proteins assists in the alternative structure. In vivo, s is buffered by the counter- formation of these complexes by shaping DNA. These active relaxing activity ofTopoI and TopoIV. The second proteins are sometimes described as histone-like, although critical function of topoisomerases is untangling and they share no structural similarity with the eukaryotic unknotting DNA (Figure 1b). Eukaryotic TopoII and E. histones (Figure 1). Chromosome-associated proteins coli TopoIV efficiently unknot and untangle chromo- include HU, H-NS, intregation host factor (IHF) and somes, and after replication this activity allows the factor for inversion stimulation (FIS). chromosomes to segregate into daughter cells. HU is encoded by two genes, hupA and hupB, which are Although an average eukaryotic nucleus is larger than closely related to each other and to the genes encoding the an E. coli cell, nuclear DNA is even more concentrated two IHF subunits. HU is the most abundant double- than bacterial DNA. Rather than being supercoiled by an stranded DNA-binding protein in E. coli, although it also

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 3 Chromosome Structure binds RNA. Although HU shows a preference for them precisely to compartments that become the daughter structurally ‘bendable’ sequences, it binds DNA relatively cells. To control chromosomal replication temporally, and nonspecifically, forming complexes that stabilize negative to integrate DNA metabolism with other aspects ofthe cell supercoils in double-stranded DNA (Figure 1). In phage Mu growth such as membrane synthesis and cell wall expan- transposition reactions, HU binds to a flexed DNA sion, E. coli makes a master regulator, the DnaA protein, structure called a transpososome with high affinity and which controls the onset ofreplication at a site called oriC. specificity. The bacterial replication cycle can be described by H-NS is a second relatively nonspecific DNA-binding progression through four stages. Less detail is available for protein that forms dimers, tetramers and possibly higher- eukaryotes, but many points are similar. As cells grow, the order associations when bound to DNA. Like HU, H-NS mass ofprotein and membrane increases to a critical point constrains negative supercoils. H-NS binds ‘bendable’ that triggers the initiation ofreplication. Two replisomes DNA, which include AT-rich sequences often found near are associated in a ‘factory’ that moves to the cell centre bacterial promoters; H-NS also modulates the transcrip- during chromosomal elongation. As DNA strands are tion ofnumerous operons in E. coli and oflysogenic pulled to the centre, replicated sister chromosomes migrate phages. toward opposite cell poles. Completion ofDNA synthesis FIS protein forms homodimers and binds DNA at occurs as the DNA that is pulled into the factory reaches specific sites. FIS was discovered nearly simultaneously in the terminus. At this point, chromosomes are tangled two related site-specific recombination systems: the Hin together (catenated), and all physical connections must be inversion reaction of Salmonella typhimurium and the Gin removed to allow final separation. When physical separa- inversion reaction ofphage Mu. FIS concentration rises in tion is complete, cell wall synthesis forms two daughter early log phase when the protein has regulatory roles in cells. replication initiation at oriC and transcription ofriboso- mal RNA operons, and then FIS abundance declines in stationary phase. IHF protein is composed oftwo subunits (encoded by Initiation the himA and hip genes) that are about 100 amino acids long and which make stable heterodimers. IHF protein DNA replication starts at oriC near position 2 85 ofthe binds to a consensus sequence that was first discovered in standard E. coli genetic map (Figure 2). Initiation involves phage l, where it stimulates insertional and excisional the DnaA protein-assisted assembly oftwo replisomes, recombination in vitro by a factor of 104. IHF bends DNA each dedicated to replicate halfa chromosome. Replisome severely (more than 908); such bends are critical for site- components include: the DnaB helicase, which is an ATP- specific recombination in l, for repression and activation dependent ring that moves along a single strand breaking ofphage Mu, and forrepression–activation in a number of open the double helix for polymerase access; a dimeric cellular operons, including ilv, oxyR and nifA. DNA polymerase molecule with accessory cofactors; and a In the eukaryotic nucleus, enzymes such as RNA complex called the primosome that triggers RNA-primed polymerase gain access to DNA, which remains histone initiation ofnew DNA strands on one side ofthe growing bound throughout most biochemical transactions. The fork. Each replisome has a pair of polymerase subunits: way in which multiple proteins interact with nucleosome- one synthesizes DNA continuously along the strand with coated DNA is not completely understood; however, 5’–3’ polarity, and the other subunit synthesizes DNA protein access to DNA is modulated by histone structure. discontinuously in small fragments called Okazaki pieces. Histone phosphorylation and acetylation change chroma- Thus DNA replication is semidiscontinuous. tin structure. Genes that are transcribed contain nucleo- In eukaryotic replication, initiation steps are not as fully somes with acetylated subunits ofhistones H3 and H4, understood as they are in E. coli. Initiation occurs at ars whereas inactive genes are bound by histones lacking sites, so called because they are autonomously replicating actetyl groups. In addition to the histones, eukaryotic sequences (Figure 3). Initiation is controlled by a group of chromosomes contain regulatory proteins that are much proteins called ORC (origin replication complex). Typi- less abundant. One class ofproteins, the high mobility cally, a chromosome has many ORC-binding sites, and group (HMG) family of DNA-binding proteins, bends bidirectional semidiscontinuous forks move out from DNA much like bacterial proteins IHF and FIS. several ars sites on each chromosome until they converge with other forks. Eukaryotic replication also occurs in ‘factories’, and most of the chromosome is replicated in a semiconservative and semidiscontinuous mode, as in E. DNA Replication coli. The eukaryotic replication fork behaves very similarly to that found in E. coli. The central problem in chromosome replication is generating two high-fidelity DNA copies and distributing

4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net Chromosome Structure

Continuous strand Semiconservative and semidiscontinuous replication

Replisome (a) Discontinuous strand

Escherichia coli replication

Replichore I Replichore I

oriC oriC

E. coli E. coli Watson strand template terE Crick strand template

terD terA terC terB dif terF Replichore II Replichore II

(b)

Figure 2 (a) A replication fork is shown with a replisome as a blue box, moving and synthesizing DNA from left to right. Replication is semiconservative, meaning that a new strand is synthesized on each of the two parental template strands, and semidiscontinuous, meaning that the strand that follows the fork with 5’ to 3’ polarity is made as one continuous piece while the strand that follows with overall 3’ to 5’ polarity is made discontinuously as Okazaki pieces. (b) The two circular chromosomes show the products of bidirectional replication. Initiation starts at oriC ( 2 85) and proceeds clockwise in the zone called replichore I and counterclockwise through the zone called replichore II. Replication forks meet at a terminus near the dif site ( 2 34). Strands made in the continuous mode are shown in red, and discontinuous strands in blue. Also included in the map are the seven ribosomal RNA operons (arrowheads), five of which reside in replichore I and two of which are found in replichore II, six ter sites and the dif site. Note that the two daughters are synthesized with different semidiscontinuous styles in replichores II and I so that the Watson and Crick strands are isomers.

Elongation Because replication is semidiscontinuous, the sister chromosomes (shown at the bottom of Figure 2) are After initiation, duplication of the entire 4.6-Mbp E. coli replication isomers. The template strand with 5’ to 3’ chromosome takes 40 min. One replisome copies DNA in polarity in the clockwise direction (the Watson strand the clockwise direction (replichore I), while the other template in Figure 2) always contains DNA replichore I, replisome copies DNA counterclockwise (replichore II) which is made discontinuously, and DNA replichore II, (Figure 2). DNA polymerase synthesizes new strands at a which is made continuously; the Crick strand template staggering rate of300 nucleotides per second. Replication always has the opposite style. Mutation and recombina- is semiconservative, meaning that one newly synthesized tion rates differ between discontinuous and continuous strand is made to match each parental template. Because replication modes, so the sisters are not always equivalent. DNA polymerases synthesize DNA only in the 5’ to 3’ As replication proceeds, positive supercoils build up in direction, and because replication forks copy both front of the fork, and the daughter chromosomes become templates at nearly the same time, there is asymmetry in entangled behind the fork (Figure 4). These two topological the mechanism ofchain growth. One strand is made as a problems are solved in similar ways in the two kingdoms. single piece while the other strand is synthesized in a series For E. coli, DNA gyrase removes positive supercoils ahead of1-kb segments, with each segment being initiated with a ofthe fork,and TopoIV removes the links between the small RNA molecule. These are called Okazaki pieces, and daughter chromosomes, which are called catenanes. they are stitched together after removal of the RNA Eukaryotic TopoI eliminates the positive supercoils primers. This semidiscontinuous pattern ofDNA replica- formed ahead of the fork, and TopoII decatenates the tion is a recurring theme in organisms as diverse as plants chromosomes for segregation (Figure 4). and humans.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 5 Chromosome Structure

Telomere TopoIV Escherichia coli Gyrase

+ Supercoils + Catenanes Watson Crick

TopoIIEukaryotes TopoI Ars1

Figure 4 Topology of DNA replication. Movement of a replication fork produces positive supercoiling ahead of the fork and results in entanglements of the sister chromosomes, called catenanes, behind the fork. Positive supercoils are removed by gyrase in bacteria and by TopoI in eukaryotes, whereas TopoIV resolves catenanes in bacteria and TopoII in eukaryotes.

replicated by a special DNA polymerase called telomerase, which is related to the reverse transcriptase ofretroviruses. Telomerase synthesizes a simple repeat sequence that is added on to every chromosome using an RNA template Centromere that is part ofthe enzyme. In most organisms telomerase is not expressed after cell differentiation, and consequently the telomere sequences shorten with age, eventually causing cell senescence and death.

Termination In E. coli, replication is completed in a region ofthe chromosome called the terminus, which is 1808 around the circular genetic map from oriC. Two special sites are found Ars2 near the terminus. First, the terminus is surrounded by at least six ter sites (Figure 2, blue boxes) which bind the Tus protein. A ter–Tus complex induces replication forks to stop or pause in a unidirectional fashion by impeding the movement ofDnaB helicase. Ter sites work in only one orientation, and they are ordered so that replication forks can proceed to the terminus but not continue in the direction opposite to normal fork movement. Thus, ter sites ensure that replication produces no more than one round ofsynthesis per initiation event. The second special site at the terminus is called dif, which functions to monomerize dimeric chromosomes during segregation (see Telomere below in site-specific recombination). The processes involved in terminating eukaryotic replication forks are Figure 3 Structure and replication pattern of a eukaryotic chromosome. Eukaryotic chromosomes are linear structures which have special structures not yet known. at each end called telomeres (green) and an organizer centre called the centromere which attaches the chromosome to the spindle during chromosome segregation. Replication is initiated at ars sites, and Segregation replication is carried out semidiscontinuously so that the two strands are replication isomers. At the conclusion ofreplication ofa circular chromosome, all physical barriers must be eliminated before daughter chromosomes completely separate. Replication leaves One segment ofa eukaryotic chromosome that is sister chromosomes interlocked by multiple catenane links different from prokaryotic chromosomes is the tip of the (Figure 1). To uncuff the DNA molecules, both gyrase and chromosome, the telomere (Figure 3). Telomeres are DNA topoisomerase work together. Once chromosomes

6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net Chromosome Structure separate, cell wall synthesis can form a permanent barrier changes occur: each pair ofreplicated chromosomes moves between daughter cells. to the cell centre and then the nuclear membrane begins to Bacteria can constrict the cell cycle to allow doubling dissolve. In stage 3, anaphase, one centromere ofeach pair times that are shorter than the 40-min period necessary to ofchromosomes is attached to a set offibres called the make one complete chromosomal copy. Under conditions spindle, and molecular motors pull one ofeach chromo- ofrapid growth, initiation at oriC speeds up so that some pair to opposite cell poles. In stage 4, telophase, the daughter cells inherit one fully replicated chromosome plus nuclear membrane is reformed and daughter cells are two or more partially replicated chromosomes at the time separated by the synthesis ofa new septum. ofcell division (dichotomous replication).

Transcription Eukaryotic Segregation A fundamental problem in biology is understanding how Following DNA replication in eukaryotes, which occurs in organisms respond to the wide variety ofenvironmental a part ofthe growth cycle called the S phase, cells move conditions that a cell or a population ofcells encounters. through a mechanical cycle called mitosis to distribute the As noted above, different bacterial species grow over an replicated chromosomes to each daughter cell (Figure 5). amazing range oftemperatures and environmental niches, Mitosis proceeds through four stages. The first is the but even a single organism can adapt to widely varying prophase in which, after replication, each chromosome growth states, from aerobic growth on rich nutrients to becomes condensed. Stage 2 is metaphase, where two anaerobic growth in minimal salts and a single carbon and

Prophase

S phase Metaphase

Telophase Anaphase

Figure 5 Chromosome segregation in eukaryotes is completed in the mitotic cycle. Mitosis proceeds through four stages, prophase, metaphase, anaphase and telophase, as described in the text.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 7 Chromosome Structure nitrogen source. One key to efficient growth is control of transcribing RNA polymerase molecule moving opposite gene expression at the level ofRNA transcription. The to the direction ofreplication forkmovement. In both process ofmaking ofan RNA complement to a DNA gene cases, the replisomes can pass RNA polymerases without is called transcription. causing them to release transcripts or dissociate from To accommodate complex regulatory scenarios, chro- DNA. However, most highly transcribed genes are mosomes are divided into discrete transcriptional modules transcribed in the same direction as replication forks called operons. An operon has two functional components: move. This includes all seven ofthe ribosomal RNA the operator (or control region) and the coding sequence, operons (black arrowheads in Figure 2b) and 53 ofthe 86 which encodes the protein or RNA product. Although transfer RNA genes. early studies focused on operons such as lac, trp and his, In eukaryotic chromosomes, the RNA polymerase which produce several proteins from a single operator, responsible for transcribing most genes is remarkably 73% ofthe 2600 different E. coli operons encode only one similar at the structural level to the E. coli RNA protein. The operator includes at least one binding site for polymerase. However, regulatory mechanisms are differ- RNA polymerase, which is the enzyme that makes a ent. Eukaryotic genes have a region called the promoter complementary RNA copy ofDNA. RNA polymerase has which is where RNA polymerase binds and starts a core subunit, plus a specificity factor called sigma (s). transcription. However, polymerase binding and its ability Sigma factors direct RNA polymerase to bind a specific to initiate transcription is influenced by sites called subset ofpromoters. In E. coli there are four sigma factors: transcription enhancers that can be upstream or down- s70, which controls many housekeeping genes and most stream ofthe promoter (relative to the direction of intermediary metabolism operons; sE, which is specific for transcription). Enhancers act over very large distances, nitrogen fixation genes; sS, which controls genes that are and so DNA looping is required to bring enhancers into turned on in stationary phase; and fliA, which is specific for contact with RNA polymerase at promoters. In addition to flagellar genes. enhancers, there are proteins called co-activators that must In addition to RNA polymerase, promoters contain bind to RNA polymerase to stimulate transcription. While both positive and negative regulatory proteins. Negative there are many questions about how co-activators work, regulatory proteins are called repressors, and they block one important known function is to modify histones by transcription by either impeding access ofRNA polymer- acetylating the H3 and H4 subunits. ase or by inhibiting its ability to initiate RNA chains. The LacI repressor blocks expression ofthe lactose genes when no lactose is available for metabolism. There are also positively acting proteins called activators, which stimulate Recombination RNA polymerase transcription, either by stabilizing its binding to a promoter or by stimulating the ability ofRNA Chromosomes undergo three types ofgenetic recombina- polymerase to initiate RNA chains. One example ofa well- tion: homologous recombination, site-specific recombina- characterized activator protein is the cyclic adenosine tion and transposition. Homologous recombination monophosphate (cAMP)-binding protein (CAP), which involves exchanges between regions ofidentical or nearly binds to a large number ofoperator sites and stimulates identical sequences that are 300 bp or longer, with the transcription when cAMP levels are high. efficiency ofrecombination increasing up to several kbp. In bacteria, replication and transcription can occur Several repetitive sequences, including the seven copies of simultaneously. Replication requires all attached, double the ribosomal RNA operon in the E. coli genome, are large strand-specific DNA-binding proteins to be temporarily enough to stimulate efficient recombination (Figure 2b). In displaced during complementary strand unwinding and E. coli, the pathway ofhomologous recombination is synthesis. The proteins that have to be removed include the carried out and regulated by four genes: recA, recB, recC chromosome-associated proteins (see section on Chroma- and recD. Homologous recombination is responsible for tin above), all repressors and operon activator proteins, the introduction ofnew informationinto the bacterial and the RNA polymerase. Transcription presents a special genome through mechanisms oftransformation,conjuga- problem for the replisome. Because transcription and tion and transduction. However, another important replication occur simultaneously, two situations arise function of recombination is DNA repair. Recombination where transcription machinery and replication machinery allows replication forks that have stalled or fallen apart to collide. One case is where transcription and replication restart DNA synthesis, in addition to allowing one move in the same direction. Because a replisome pulls in damaged chromosome to be repaired using information DNA at 500 bp per second and RNA polymerase from a sister chromosome. Thus, in addition to being an transcribes DNA at 50 bp per second, replication forks agent ofchange, recombination helps chromosomes rapidly overtake RNA polymerase, even when both remain the same. enzymes are headed in the same direction. The more Homologous recombination is critical for repair of striking encounter occurs when DNA replication meets a DNA damage caused by chemical modification by

8 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net Chromosome Structure alkylating agents, by ultraviolet light, and X-ray-induced protein called the transposase, which binds the ends and chromosome breaks. DNA damage is also caused by stimulates transposon movement. The chromosome of E. internal oxygen metabolism, which generates free radicals. coli has 42 different transposons that have invaded by Replication errors also cause DNA breaks. The RecABCD horizontal transfer from other bacteria or viruses. pathway corrects a damaged copy ofa chromosome by In eukaryotes, transposons (usually called retrotran- using identical sequences in a sister chromosome in the sposons because oftheir similarity to retroviruses and their same cell. As noted above, when rapid growth is underway, dependence on reverse transcriptase for replication) make cells have more than one chromosome. RecA protein up a large fraction of total chromosomal DNA. In human serves two roles in repairing chromosome damage. First, it cells, sequences called short interspersed nuclear elements binds to single-stranded regions ofa broken chromosome (SINEs), which are about 300 bp long are present in about and facilitates a search for the homologous sequence, and 106 copies and represent 5% ofthe mass ofDNA in a then forms a hybrid molecule that starts recombination. haploid genome. One particular SINE, called AluI, is RecA also regulates gene expression. When DNA damage present on average once every 5000 bp in every human occurs, RecA protein inactivates a repressor called LexA, chromosome. There are also long interspersed nuclear which results in the expression ofover a dozen DNA repair elements (LINEs) ofabout 6 kb that are present in about proteins. After DNA repair is complete, RecA protein 105 copies and represent 15% ofthe haploid chromosomal stops inactivating LexA repressor and DNA metabolism mass. What function, if any, these sequences provide for returns to normal. the host organism is questionable, but many genetic Recombination is a critical repair pathway in mamma- mutations have been attributed to gene disruption caused lian chromosomes as well. Proteins that carry out by recent transposon insertions. biochemical reactions similar to the E. coli RecABC system have been identified. A protein called p53 coordinates the activity ofmany DNA repair proteins. Repair enzymes are stored at chromosome telomeres, and Site-specific Recombination after a signal from p53 these proteins migrate to sites of DNA damage to restore chromosome function. Mutations Site-specific recombination provides an efficient mechan- in DNA repair genes have been discovered to be ism for rearranging DNA at sites less than 100 bp in length responsible for several human genetic syndromes that (below the level required for homologous recombination). result in premature ageing and high spontaneous rates of Site-specific recombination systems cause insertion and cancer. excision ofdifferent lysogenic viruses, inversion ofregions flanked by inverted sites, and deletions when two sites are directly repeated in a chromosome. Bacteriophage l contains the best-studied site-specific recombination sys- Chi Sites tem – the Int/Xis system – which allows a prophage to integrate into the bacterial chromosome at one point called One sequence in E. coli influences the efficiency and attB, or subsequently to excise and replicate in the lytic direction ofrecombination: the chi sequence, mode. Site-specific systems use a protein recombinase to GCTGGTGG. A chi site stimulates recombination direc- bind and recombine short sequences, usually about 20 bp tionally, by triggering the pairing ofchromosome se- long. quences near chi. Like ter sites in replication, chi sites act in One site-specific recombination system in E. coli plays a only one direction. About 75% ofall chi sites are arranged crucial role in chromosome segregation. DNA synthesis in the clockwise direction in replichore I and counter- may generate breaks (often on the discontinuous strand) clockwise in replichore II, so that they instruct recombina- that stimulate recombination between daughter chromo- tion to proceed according to the direction ofDNA somes. Ifan odd number ofcrossovers occurs between replication. daughters, the chromosomes will be dimerized at the completion ofDNA replication. In E. coli, about 15% of the normal replication cycles produce dimeric chromo- Transposons somes. The site-specific recombination system that resolves these molecules is composed ofthe dif site at the terminus Transposons are discrete genetic elements that move from (Figure 2) and recombination proteins XerC and XerD, one site or from one chromosome to another, often with which efficiently separate daughter chromosomes just little regard to any specific DNA target sequence. before cell division. Transposons sometimes carry with them genes for In eukaryotes, site-specific recombination also has many resistance to antibiotics such as tetracycline (Tn10) and critical roles in biological development. One spectacular kanamycin (Tn5). A transposon generally has inverted example is the mammalian immune system, where different repeats at the ends ofthe element, and encodes at least one chromosomal segments called V, D and J genes are cut and

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 9 Chromosome Structure spliced together to produce antibodies. The mammalian systems designed to promote chromosome stability is immune system can make antibodies to a diverse array of nearly equal to the forces that promote change. different structures by cutting and pasting genes together in an immense number ofdifferent ways. The antibody system is essential for life, because it protects an organism from invasion by bacterial and viral pathogens. Thus, site- Further Reading specific recombination has evolved as one key mechanism Casjens S (1998) Bacterial genome structure. Annual Review ofGenetics that changes the genome in a small subset ofdifferentiated 32: 339–377. cells, and this ultimately allows an organism to survive and Charlebois RL (ed) (1999) Organization ofthe Prokaryotic Genome . stay the same. Washington DC: ASM Press. With multiple transposons, efficient homologous and Cozzarelli NR and Wang JC (1990) DNA Topology and its Biological site-specific recombination systems, the occurrence of Effects. Cold Spring Harbor, New York: Cold Spring Harbor Press. frequent chromosome breaks, and the presence of multiple Kornberg A and Baker T (1991) DNA Replication, 2nd edn. New York: long inverted and direct repeats, it would seem impossible WH Freeman. Kuzminov A (1999) Recombinational repair ofDNA damage in for chromosomes to remain constant through time. Escherichia coli and bacteriophage l. Microbiology and Molecular Surprisingly, the genetic maps of E. coli and S. typhimur- Biology Reviews 63: 751–813. ium are very similar after 140 million years of separation Neidhardt FC (ed.) (1996) Escherichia coli and Salmonella. Washington from a common ancestor. Thus, the efficiency of enzyme DC: ASM Press.

10 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net