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Chapter 30: DNA Replication, Repair, and Recombination

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Chapter 30: DNA Replication, Repair, and Recombination Chapter 30: DNA Replication, Repair, and Recombination 1. DNA Replication: An overview 2. Enzymes of Replication 3. Prokaryotic Replication 4. Eukaryotic Replication 5. Repair of DNA 6. Recombination and Mobile Genetic Elements 7. DNA Methylation and Trinucleotide Repeat Expansion DNA Replication • DNA double strand -> template for duplication, Replication • Chemically similar to transcription • As complex as translation but enzymes in only few copies/cell • Extremely accurate: 10-10 mistakes/base • Extremely regulated: only once per cell division Action of DNA polymerase • Template • dNTPs • 5’ -> 3’ direction • Semi-conservative Replication of DNA Unwinding of dsDNA: - Rate in E. coli: 1000nt/sec - 100rev/sec (10bp/turn) Negative supercoils by: DNA Gyrase type II topoisomerase, ATP E. coli theta replication • autoradiogramm • branch point called “replication fork” • unidirectional / bidirectional • prokaryotes and bacteriophages have only one origin of replication Unidirectional vs. bidirectional θ replication [3H]tymidine pulse-labelling Semidiscontinuous DNA replication Okazaki fragments: Discontinous ! 1000-2000nt in prokaryotes 100-200nt in eukaryotes Joined by DNA ligase Replication eye in Drosophila melanogaster DNA Priming of DNA synthesis by short RNA segments E. coli: RNA Polymerase Primase, rifampicin sensitive Removal of RNA primers 2. Enzymes of replication DNA Replication requires (in order of appearance): 1. DNA Topoisomerase 2. Helicases 3. ssDNA binding proteins 4. RNA primer synthesis 5. DNA polymerase 6. Enzyme to remove RNA primers 7. Link Okazaki fragments E. coli DNA polymerase I in complex with a dsDNA Arthur Kornberg, 1957 DNA Polymerase I 5’->3’ synthesis Processive, 20nt Recognizes dNTP based on base pairing Right hand sructure Editing activity: 3’->5’ exonuclease 5’->3’ exonuclease (proofreading) Fidelity 10-7 Klenow fragment Lacks 5’->3’ exo, lacks N- term. Nick translation as catalyzed by Pol I Used to radiolabel DNA probes for Southern/Northern DNaseI, αP32dNTP Pol I functions to repair DNA E. coli, Pol I mutant are viable but sensitive to UV and chemical mutagens Essentisl physiological function of Pol I 5’->3’ exonuclease is to excise RNA primers, role in replication DNA Polymerase III Pol III is replicase of E. coli Holoenzyme consists of more than 10 subunits β subunit confers processivity >5000nt β subunit form a ring like sliding clamp with 80Å diameter hole, sliding clamp/ β clamp Properties of E. coli DNA Polymerases Components of E. coli DNA Polymerase III Holoenzyme β subunit of E. coli Pol III holoenzyme Unwinding of DNA 3 proteins required to advance replication fork: Helicase, DnaB, hexameric, ATP-dep., 5’->3’,AAA+ Strand separation, Rep helicase, dimer, ATP-dep. ssDNA binding protein, prevent re-annealing, tetramer Unwinding and Binding Proteins of E. coli DNA Replication Active, rolling mechanism for DNA unwinding by Rep helicase DNA ligase Ligating single strand nicks between Okazaki fragments E. coli: NAD-dependent T4 phage, ATP-dependent blunt end ligation Primase Synthesis of RNA primers fro Okazaki fragments: 5’->3’ In vitro 11nt ±1 Prokaryotic Replication Bacteriophages Coliphages: M13, φX174 M13: 6408nt ssDNA(+), circular Replication->RF Leading strand synthesis φX174 Replication 5386nt ssDNA circular Replication more complex than M13 Requires primosome Paradigm for lagging strand synthesis 6step process a. coating b. primosome assembly c. migration d. priming e. Pol III extension f. Pol I removes primers g. ligation, supercoiling Micrograph of a primosome Proteins of the Primosomea The rolling circle mode of DNA replication a. Specific cut at + strand b. Extension of + strand c. Tandem-linked + strands d. Separation by endonuclease e. packaging Rolling circle = Sigma replication φX174 (+) strand replication by the looped rolling circle mode φX174 (+) strand synthesis as model for leading strand replication 1. Cut by gene A protein 2. Pol II extension 3. Cut + ligation The replication of E. coli DNA Bidirectional, theta replication leading and lagging strand synthesis occurs on a common 900kD multisubunit particle: the replisome -> loop of lagging strand Initiation: at oriC, 245bp segment The replication of E. coli DNA A model for DNA replication initiation at oriC oriC, 245bp segment Contains 5 DnaA boxes Melting, P1 Penicillium citrinum endodunclease Specific for ssDNA Prepriming complex (DnaB DnaC)6 Initiation of DNA replication is strictly regulated Only 1 replication/cell cycle Doubling time 20min-10h 1000nt/sec 4.6 106bp genome -> 40min/replication -> multiforked chromosomes Sequestration of hemimethylated oriC Electron micrograph of an intact and supercoiled E. coli chromosome attached to two fragments of the cell membrane Schematic diagram of the clamp loading cycle β clamp responsible for high processivity of Pol III Must be “loaded” onto DNA by a clamp loader ATP-dep. AAA+ Termination of replication Large 350 kb region in E. coli genome Flanked by 7 nonpalindromic nearly identical termination Sites Replication fork counterclockwise passes through TerG,F, B, and C but stops at TerA Analogous for other direction Ter act as valves Ter-action requires binding of Tus protein Without Ter, collision of replication forks terminates Fidelity of Replication Complexity of replication (>20 proteins) important for high fidelity: T4 phage reversion 10-8 - 10-10 High accuracy due to: 1. Balanced dNTP levels 2. Polymerase reaction itself, pairing 3. 3’->5’ exonuclease of Pol I and Pol III 4. Repair systems -> see later Why only 5’->3’ synthesis ? 3’->5’ extension would require retention of 5’ triphosphate This would be lost upon editing ! Eukaryotic Replication Remarkable degree of similarity to prok. replication But linear chromosomes -> ends ? Cell cycle regulation, can last 8h to > 100 days Most variation in G1 phase/Go phase Irreversible decision to proliferate is made in G1 Checkpoint Controlled by cyclins and cyclin-dep. kinases Best understood from yeast (budding, fission) The eukaryotic cell cycle Eukaryotic cells contain many polymerases 6 families: A, E. coli Pol I, Pol γ (mitochondrial) B, E. coli Pol II, Pol α, Pol δ C, E. coli Pol III D, X, Y Pol δ, unlimited processivity when in complex with PCNA, proliferating cell nuclear antigen (systemic lupus erythematosus), β clamp function Properties of Some Animal DNA Polymerases Structure of PCNA Eukaryotic chromosomes consist of numerous replicons Multiple replication origins, every 3-300kb Replication rate 50nt/sec, 20x slower than E. coli But 60x more DNA Replication would require 1 month Clusters of 20-80 adjacent replicons Not simultaneously, but ensure they initiate only once Assembly of the initiator complex in 2 stages To prevent multiple rounds of initiation: Assembly of pre-RC in G1 phase (licensed) Activation at S phase Origin can “fire” only once Origin = ARS (autonomously replicating sequences) Re-replication prevented by Cdks and Geminin ORC, origin recognition complex Hexamer, Orc1-Orc6 (DnaA analog) MCM, minichr. maintenance funct. Removal of RNA primers 2 enzymes: RNase H1, removes most of the RNA leaving a single 5’ ribonucleotide (H, hybrid) Flap endonuclease-1 (FEN1) removes single single 5’ ribonucleotide Mitochondrial DNA is replicated in D- loops 15kb circular genome Leading strand synthesis precedes lagging strand Leading strand forms displacement loop (D-loop) Reverse transcriptase Retroviruses: RNA containing eukaryotic viruses, e.g. HIV Replicate from RNA genome Copy RNA into DNA by Reverse Transcripase (RT) Similar to Pol I, 5’->3’ synthesis of DNA from RNA template, primed by host tRNA RNA is degraded by RNase H ssDNA directs dsDNA synthesis dsDNA integration into host genome RT: important tool for cDNA synthesis, oligo-dT primed Reverse transcriptase Structure of HIV-1 reverse transcriptase RT inhibitors Telomers and Telomerase How are the ends of linear chromosomes replicated ? Problem: no priming at 5’ of lagging strand possible without shortening of the chromosome upon every replication Telomer sequence: unusual, G-rich, 3’ overhang (20-200bp) Specialized enzyme: telomerase adds G-rich repeats without teplate, is ribonucleoprotein, RNA acts as template Synthesis of telomeric DNA by Tetrahymena telomerase Telomers must be capped Without telomerase, chromosome would shorten 50-100nt with every cell division Exposed telomeric ssDNA must be protected by capping with proteins, Pot1 Telomere length correlates with aging Primary cells in culture die after 20-60 divisions Such somatic cells have no telomerase activity -> Telomers shorten with every division Telomerase is active only in germ cells Analysis of fibroblast from donors of different age: No correlation with numbers of doublings in culture But correlation of telomer length with numbers of doublings Progeria: premature aging disease patients have short telomers Cancer cells have active telomerase Why do somatic cells down regulate telomerase ? Senescence may be a mechanism to protect from cancer All immortal cells express telomerase Telomeric DNA can dimerize via G-quartets Telomers form T-loops Repair of DNA DNA is not inert UV radiation, ionizing radiation, toxic chemicals, oxidative metabolism can harm DNA Spontaneous hydrolysis of 10’000 glycosidic bonds in every cell every day.... Human genome 130 genes dedicated to DNA repair Chemically similar in E. coli Chemical damage of DNA Oxidation Hydrolysis Methylation Direct reversal of damage Pyrimidine dimers are split by photolyase: UV (200-300nm) promtes Formation of cyclobutyl ring
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