1. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E DNA Replication Eukaryotes

2. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E New experimental systems gap in understanding eukaryotic replication Mutant yeast cells helpful –Applicable to mammals

3. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

4. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

5. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.19

6. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

7. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

8. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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?

9. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E In mammals human  -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

10. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.20

11. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

12. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.21

13. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

14. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

15. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Eukaryotes Okazaki fragments are smaller (~150 nucleotides) Like E. coli DNA polymerase III… –eukaryotic DNA polymerase  is dimer –suggests 1 replicative complex (replisome)

16. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 5 different DNA polymerases 3 involved in replication; 2 are not  &  Polymerase  - tightly bound to primase –initiate synthesis of each Okazaki fragment –after primase lays down short primer –polymerase  adds several bases

17. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Polymerase  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  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

18. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.22

19. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Polymerase  polymerase  -primase complex replaced by PCNA- polymerase  completes Okazaki fragment synthesis RNA primer removed & gap filled by polymerase  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

20. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Other Polymerases –Polymerase  - replicates mitochondrial DNA –Polymerase  - functions in DNA repair –Polymerase  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

21. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Polymerases Several other DNA polymerases ( ,  &  ) –specialized for replicating damaged DNA –error-prone? All polymerases –synthesize 5' —> 3' –all require primer –Polymerases ,  &  have a 3' —> 5' proofreading exo

22. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

23. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.23

24. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

25. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.25b