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DNA Replication

DNA Replication. Eukaryotes. New experimental systems. gap in understanding eukaryotic replication Mutant yeast cells helpful Applicable to mammals. New experimental systems. In vitro systems Frequently viral SV40 particularly useful Single replication origin

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DNA Replication

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  1. DNA Replication Eukaryotes Karp/CELL & MOLECULAR BIOLOGY 3E

  2. New experimental systems • gap in understanding eukaryotic replication • Mutant yeast cells helpful • Applicable to mammals Karp/CELL & MOLECULAR BIOLOGY 3E

  3. New experimental systems • In vitro systems • Frequently viral • SV40 particularly useful • Single replication origin • Site for the binding of large T antigen • initiates replication & acts as the helicase • rest of replication depends on host cell machinery Karp/CELL & MOLECULAR BIOLOGY 3E

  4. Initiation of replication in eukaryotic cells • subject to much regulation • much more DNA than bacteria • replicate their genomes in small portions (replicons) • 15 - 100 µm in length (50 - 300 kb) • each has its own bi-directional origin • subject to regulation • nearby replicons tend to replicate simultaneously • early replicons & late replicons • timing depends on gene activity or state of compaction Karp/CELL & MOLECULAR BIOLOGY 3E

  5. Figure 13.19 Karp/CELL & MOLECULAR BIOLOGY 3E

  6. Initiation of replication in eukaryotic cells • Tightly compacted heterochromatin • last regions to be replicated • heterochromtic X replicates very late in S phase • euchromatic X chromosome replicates earlier • control: once and only once each cycle Karp/CELL & MOLECULAR BIOLOGY 3E

  7. Yeast origins of replication • Called autonomous replicating sequences (ARSs) • ~400 ARSs scattered throughout yeast chromosomes • several distinct elements • Core element has conserved 11 bp sequence • specific binding site for origin recognition complex • ORC remains bound to origin throughout cell cycle • initiation triggered by binding of other proteins to ORC Karp/CELL & MOLECULAR BIOLOGY 3E

  8. In mammals • replication at a number of sites • Any type of purified, naked, vertebrate DNA works • makes study of replication origins difficult • might not possess specific ARSs • different in vivo? • chromatin structure & nucleosome positioning? Karp/CELL & MOLECULAR BIOLOGY 3E

  9. In mammals • human b-globin gene cluster rep origin • transplantable • Deleted in patients with hemoglobin Lepore syndrome • DNA is still replicated by another fork • Origin usage pattern is flexible Karp/CELL & MOLECULAR BIOLOGY 3E

  10. Figure 13.20 Karp/CELL & MOLECULAR BIOLOGY 3E

  11. In yeast • Licensing factors attracted by ORC • 6 related Mcm proteins (Mcm2 – Mcm7) • Mcm proteins loaded with the aid of Cdc6 • Occurs soon after mitosis has completed • Nuclease protection increases • DNA footprint expands with Cdc6 & Mcm • Homologs in frogs & mammals • Suggests conservation among eukaryotes Karp/CELL & MOLECULAR BIOLOGY 3E

  12. Figure 13.21 Karp/CELL & MOLECULAR BIOLOGY 3E

  13. In yeast • Activation • Mcm proteins move with the replication fork • Mcm2-Mcm7 make ring-shaped complex • Strong candidate for helicase (like DnaB in E. coli) • The fate of the Mcm proteins variable • In yeast, displaced from chromatin & exported from nucleus • In mammalian cells, displaced from DNA but remain in nucleus • Mcm’s cannot reassociate until next cell cycle Karp/CELL & MOLECULAR BIOLOGY 3E

  14. Replication requires • Helicases • single-stranded DNA-binding proteins • topoisomerases • primase • DNA polymerase • DNA ligase • Endogenous helicase not identified with certainty • In vitro studies: SV40 large T antigen = helicase Karp/CELL & MOLECULAR BIOLOGY 3E

  15. Eukaryotes • Okazaki fragments are smaller (~150 nucleotides) • Like E. coli DNA polymerase III… • eukaryotic DNA polymerase d is dimer • suggests 1 replicative complex (replisome) Karp/CELL & MOLECULAR BIOLOGY 3E

  16. 5 different DNA polymerases • 3 involved in replication; 2 are not • a, b, g, d & e • Polymerase a - tightly bound to primase • initiate synthesis of each Okazaki fragment • after primase lays down short primer • polymerase a adds several bases Karp/CELL & MOLECULAR BIOLOGY 3E

  17. Polymerase d • assembles leading strand & most of lagging strand • thought to be primary replicative enzyme • requires "sliding clamp" structure (PCNA) like polymerase III in E. coli • Sliding clamp similar to b subunit of E. coli polymerase III • In eukaryotes, it is called PCNA - proliferating cell nuclear antigen • antigen that reacts with auto-antibodies in serum of lupus erythematosus patients • clamp loader called RFC Karp/CELL & MOLECULAR BIOLOGY 3E

  18. Figure 13.22 Karp/CELL & MOLECULAR BIOLOGY 3E

  19. Polymerase d • polymerase a-primase complex replaced by PCNA-polymerase d • completes Okazaki fragment synthesis • RNA primer removed & gap filled by polymerase d • eukaryotic DNA polymerases do not have 5' —> 3' exonuclease • primers are removed by other nucleases, RNase H1 & FEN-1 • Adjoining fragments are ultimately sealed by DNA ligase Karp/CELL & MOLECULAR BIOLOGY 3E

  20. Other Polymerases • Polymerase g - replicates mitochondrial DNA • Polymerase b - functions in DNA repair • Polymerase e • appears to play a role in nuclear DNA replication • replication cannot be finished in cells lacking this polymerase • not required for in vitro replication of SV40 DNA • exact role unknown Karp/CELL & MOLECULAR BIOLOGY 3E

  21. Polymerases • Several other DNA polymerases (h, z & i) • specialized for replicating damaged DNA • error-prone? • All polymerases • synthesize 5' —> 3' • all require primer • Polymerases g, d & e have a 3' —> 5' proofreading exo Karp/CELL & MOLECULAR BIOLOGY 3E

  22. Chromatin & replication • machinery associated with nuclear matrix • huge complex of proteins • very short, hot nucleotide precursor pulse • incorporated label (>80%) is associated with matrix • label chased from matrix into the surrounding loops • immobilized replication apparatus: “conveyer belt” • Active Forks localized within 50 - 250 sites • replication foci • ~40 forks/focus • Clustering may coordinate chromosome replication Karp/CELL & MOLECULAR BIOLOGY 3E

  23. Figure 13.23 Karp/CELL & MOLECULAR BIOLOGY 3E

  24. Chromatin & replication • eukaryotic chromosomes: histone proteins • rapid assembly of nucleosomes at fork • core histone octamer (H3H4)2 tetramer and pair of H2A/H2B dimers • (H3H4)2 tetramers remain intact • distributed randomly between 2 daughter duplexes • As a result, old & new (H3H4)2 tetramers intermixed • H2A/H2B dimers separate from one another • appear to bind randomly to the new & old (H3H4)2 Karp/CELL & MOLECULAR BIOLOGY 3E

  25. Figure 13.25b Karp/CELL & MOLECULAR BIOLOGY 3E

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