Replication

1. Chapter 20 - DNA Replication, Repair and Recombination Replication Maintain genetic info from generation to generation Rapid (before cell divides) Accurate Need to correct replication errors and repair damage Recombination: shuffle pieces of DNA

2. Semiconservative DNA replication • Each strand of DNA acts as a template for synthesis of a new strand • Daughter DNA contains one parental and one newly synthesized strand

3. Chromosomal DNA Replication is Bidirectional • Replication involves initiation, elongation, and termination. • E. colichromosome is circular, double-stranded DNA • (4.6x103 kilobase pairs, >1000 bp/sec) • Replication begins at a unique site (origin) • Proceeds bidirectionally until the two replication complexes meet (termination site) • Replisome - protein machinery for replication (one replisome at each of 2 replication forks)

4. Eukaryotic replication • Eukaryotic chromosomes are large and linear DNA molecules • Fruit fly large chromosomes ~5.0x104 kb (~10x larger than E. coli) • Multiple sites of initiation of DNA synthesis • (versus one site in E. coli) Thus, overall time frame to complete replication is the same as in E coli

5. Drosophila DNA replicating Large # of replication forks at opposite ends of “bubbles” of duplicated DNA

6. Drosophila DNA replicating Rate of fork movement is slower (chromatin structure) Larger genome size Multiple sites of initiation Thus, overall time frame to complete replication is the same as in E coli

7. DNA Polymerase • E. coli contains three DNA polymerases • DNA polymerase I - repairs DNA and participates in DNA synthesis of the lagging strand • DNA polymerase II - role in DNA repair • DNA polymerase III - the major DNA replication enzyme, responsible for chain elongation DNA polymerase III a, e,  = polymerization core b4 = two sliding clamps.  complex = assembly. 10 diff subunits DNA directed DNA polymerase III

8. DNA Polymerase Synthesizes Two Strands Simultaneously DNA polymerase III

9. DNA Polymerase III Remains Bound to the Replication Fork • DNA polymerase III is a processive (rather than distributive) enzyme (remains bound to the replication fork until replication is complete) • b-Subunits forms a sliding clamp which surrounds the DNA molecule Few Pol III needed for complete replication Allows rapid rate

10. DNA Polymerase III Remains Bound to the Replication Fork • DNA polymerase III is a processive (rather than distributive) enzyme (remains bound to the replication fork until replication is complete) • b-Subunits forms a sliding clamp which surrounds the DNA molecule Few Pol III needed for Complete replication Allows rapid rate Similar concept used in other systems Bacteriophage pol

11. Chain Elongation Is a Nucleotidyl-Group-Transfer Reaction One nucleotide at a time dNTP substrate forms base pair 3’OH nucleophilic attack onto alpha phosphate of dNTP PPi cleavage…..does what??? 5’ to 3’ direction Requires template and primer to syn DNA (3’OH)

12. Proofreading Corrects Polymerization Errors DNA polymerase III holoenzyme also possesses 3’ 5’ exonuclease activity removes mispairednucleotide before polymerization continues Recognizes distortion in the DNA caused by incorrectly paired bases Pol III can catalyze both chain elongation and degradation Polymerase error = 10-5, nuclease error 10-2; Overall error rate = 10-7 Lowest rate of any enzyme but mistakes do occur and transmitted

13. DNA Polymerase Synthesizes Two Strands Simultaneously • DNA pol III catalyzes chain elongation only in the 5’ 3’ direction (antiparallel DNA strands) • Leading strand - synthesized by polymerization in the samedirection as fork movement • Lagging strand - synthesized by polymerization in the oppositedirection of fork movement • Two core complexes of DNA pol III, one for leading, one for lagging strand

14. Replisome DNA synthesis Two core complexes of DNA pol III one for leading, one for lagging strand Replisome = primosome+ DNA Pol

15. fork fork 5’ 3’ 5’ 3’ Lagging-Strand Synthesisis Discontinuous • Leadingstrand is synthesized as one continuous polynucleotide (beginning at origin and ending at the termination site) • Laggingstrand is synthesized discontinuouslyin short pieces (Okazaki fragments) • Pieces of the lagging strand are then joined by a separate reaction • By Pol I and DNA ligase Overall the process is semidiscontinuous

16. Demonstration of discontinuous DNA synthesis • (In lagging strand) Pulse label E. coli by short time-period With 3H-dTTP

17. (From previous slide)

18. RNA Primer Begins Each Okazaki Fragment • Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template) • DNA pol III uses the RNA primer to start the lagging-strand DNA synthesis • Replisome - includes primosome, DNA pol III DNA dependent RNA pol DNA pol can’t begin de novo (needs existing 3’OH) Now need to join Okazaki fragments

19. Joining of Okazaki fragments by DNA pol I and DNA ligase Three steps: Remove RNA primer Synthesis replacement DNA Seal 2 DNA fragments

20. Single polypeptide has both activities

21. Removal of RNA primer essential DNA ligase only uses dsDNA

23. DNA ligase mechanism

24. First enzyme found to syn DNA Pol I can be cleaved into Large frag: pol and proofreading (3’-5’ exo) Small frag: 5’-3’ exonuclease • Klenow (large) fragment of DNA pol I, lacks 5’-3’ exonuclease activity Simple enzyme used to syn DNA in the test tube

25. Replisome DNA synthesis Helicase in primase unwinds DNA DNA syn coupled to unwinding parental DNA But there is no large stretch of ssDNA SSB

26. SSB keeps DNA free of 2nd structure (good template) Unwinding assisted by topoisomerases Relieve supercoiling Not part of replisome topoII: gyrase Lagging strand encounters okazaki frag Releases lagging strand

28. Initiation and Termination of DNA Replication E. coli • Replisome assembles at origin site (oriC) • Initial assembly depends on local unwinding of the DNA caused by binding certain proteins • DnaA is one initiation protein • Terminator utilization substance (Tus) binds to the ter site • Tus inhibits helicase activity and thus prevents replication forks continuing through this region

29. Eukaryotic cell cycle: regulation of replication 1N M phase: Mitotic cell division S phase: DNA replication Only replicate once: One Drosophila chrom 600 replication forks Origins only used once! During S phase 2N M-G1: ORC assembly at each Ori formation of pre-initiation complex S phase: replisomes “fire” in response to S-phase protein kinase high levels of kinase prevents re-loading of complexes After G2 the kinase is cleared and ORC assemble on new chromosome

30. DNA repair mechanisms The only cellular macromolecule that can be repaired Cost to organism (mutated or damaged DNA) far outweigh the energy of repair Repair protects individual cells and subsequent generations Single-celled organism- one mutation can kill DNA damage: base modifications, nucleotide deletions x-link DNA strands “oxygen catastrophe” introduced oxidative stress to nucleic acid system

31. DNA repair mechanisms The only cellular macromolecule that can be repaired Cost to organism (mutated or damaged DNA) far outweigh the energy of repair Repair protects individual cells and subsequent generations Single-celled organism- one mutation can kill DNA damage: base modifications, nucleotide deletions x-link DNA strands • Specific repair enzymes scan DNA to detect any alterations • Lesions may be fixed by direct repair, which does not require breaking the phosphodiester backbone of DNA or by more complicated reactions

32. Repair after Photodimerization: An Example of Direct Repair • Double-helical DNA is very sensitive to damage by UV light • Dimerization of adjacent pyrimidines in a DNA strand is common (e.g. thymines) • Replication cannot proceed in the presence of pyrimidine dimers (template strand is distorted) • Thymine dimers are repaired in all organisms

33. Repair of thymine dimers by DNA photolyase

35. Direct repair: DNA backbone not cleaved photoreactivation

36. Excision Repair • DNA can be damaged by alkylation, methylation, deamination, loss of heterocyclic bases (depurination or depyrimidization) • General excision-repair pathway can repair many of these defects • Overall pathway is similar in all organisms UvrABC endonuclease 12-13 nucleotide gap

37. Most common type of DNA damage • Hydrolytic deamination of cytosine to uracil • Uracil in place of cytosine causes incorporation of an incorrect base during replication • DNA glycosylases hydrolyze base-sugar N-glycosidic bonds • Deaminated bases are then removed and replaced Explains why uracil not part of DNA so this reaction will be recognized as an error

38. Base excision repair Uracil N-glycosylase Flips base out of helix and hydrolyzes Apurinic and apyrimidimic site AP endonuclease Repair of damage from deamination of cytosine

39. DNA repair by recombination 1. Repair 2. Exchange info Similar proteins utilized in DNA repair

40. Homologous Recombination • Recombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome • Homologous recombination - occurs between pieces of DNA that have closely related sequences • Nonhomologousrecombination occurs between unrelated sequences (e.g. Transposons ) 1. Repair 2. Exchange info

41. The Holliday Model of General Recombination E. coli proteins: Cleavage and ssDNA formation RecBCD endonuclease Strand invasion RecA Branch migration RuvA and B Resolution RuvC

42. RecA catalyzes strand exchange E. coli Recombination starts with generation of single-stranded DNA with a free 3’ end RecBCD endonuclease binds to DNA, cleaves one strand, then unwinds DNA in an ATP-dependent reaction Strand exchange begins when single-stranded DNA invades a neighboring double helix Rec A is a strand exchange protein Promote triple-strand interaction Recognize sequence homology Catalyzes strand exchange

43. Action of Ruv proteins at Holliday junctions

44. Fig 20.29 Branch migration and resolution

45. Fig 20.28 Model of RuvA and RuvB bound to Holliday junction