1. FCH 532 Lecture 12 Chapter 30: DNA replication Exams graded: average was 69

2. Table 11.1 Comparison of E. coli DNA polymerases Primary replicating enzyme in E. coli cells is thought to be DNA pol III: faster, more complex structure. DNA pol I and II probably serve in editing and repair of DNA.

3. A DNA Replication Paradox: 1.DNA strands must be copied in both directions during replication. 2.However, all known DNA polymerases catalyze chain formation in the 5’  3’ direction. Paradox resolved by Reiji Okazaki and coworkers: Observed short 1000-2000 nucleotide fragments during replication. Small fragments are covalently joined in later steps to form completed lagging daughter strand.

4. Semidiscontinous replication If DNA strands are extended 5’ to 3’, how can the antiparallel strand by simultaneously replicated past the replication fork? Okazaki used pulse-chase experiment. E. coli DNA pulse-labeled 30 s with of [ 3 H]thymidine that changes the sedimentation coefficient of newly made DNA from 7S to 11S (Okazaki fragments). Okazaki fragments: 1000-2000 nt in prokaryotes; 100- 200 nt in eukaryotes Follow [ 3 H]thymidine pulse with transfer to unlabeled medium (pulse-chase)-the resulting radioactively labeled DNA sediments at rate that increases with time the cells grow in unlabeled medium. Okazaki fragments incorporated into the larger DNA.

5. Figure 30-5Semidiscontinuous DNA replication. In DNA replication, both daughter strands (leading strand red, lagging strand blue) are synthesized in their 5¢ ® 3¢ directions. Page 1138

6. RNA Primers Need a 3’-OH group to extend DNA chain. Analysis of Okazaki fragments revealed that they have short (1-60 nt) RNA segments complementary to the template DNA. RNA primers catalyzed by 2 enzymes: RNA polymerase, large (459 kD), mediates transcription, rifampicin sensitive. Primase (DnaG), small (60 kD), rifampicin resistant. Rifampicin inhibits only leading strand synthesis, therefore, primase initiates Okazaki fragments. Leading strand can be mediated in vitro by both, is better when both enzymes present.

7. Figure 30-7Priming of DNA synthesis by short RNA segments. Page 1139

8. Figure 11.8 Closeup of a replication fork showing initiation of the continuous leading strand and the discontinuous, lagging strand (Okazaki fragments) All known DNA polymerases catalyze chain formation in the 5’  3’ direction. DNA strands must be copied in both directions!

9. Figure 11.9a Complete scheme showing sequential steps of replication process. 1.Helicase (unwinding protein / rep protein) recognizes and binds origin of replication. Catalyzes separation of the two DNA strands by disrupting H- bonding between base pairs. Endothermic reaction is coupled to hydrolysis of ATP. DNA gyrase (a topoisomerase) assists in unwinding by inducing supercoiling.

10. Figure 11.9b Complete scheme showing sequential steps of replication process. 2.Single-stranded DNA binding proteins (SSB) bind exposed strands of DNA. Protect it from hydrolytic cleavage of phosphodiester bonds.

11. Figure 11.9c Complete scheme showing sequential steps of replication process. 3.Primer synthesis: Short complementary stretch of RNA (4-10 bases) is synthesized by primase enzyme. Primer with free 3’-OH is required by DNA pol III to start 2nd strand synthesis. RNA primer is later degraded by 5’->3’ exonuclease action of DNA pol I, RNaseH enzymes.

12. Figure 11.9d Complete scheme showing sequential steps of replication process. 4.DNA synthesis by DNA pol III begins, extending leading and lagging strands. DNA synthesis continues until it meets next fragment.

13. Figure 11.9e Complete scheme showing sequential steps of replication process. 5.RNA primers are removed by 5’  3’ exonuclease action of DNA polymerase I, small gaps are filled in by DNA polymerase I.

14. Figure 11.9f Complete scheme showing sequential steps of replication process. 6.Final gap between new strands is closed by DNA ligase enzyme. Requires ATP to join 3’ OH on one fragment and 5’ phosphate on second fragment.

15. Figure 11.10 The DNA ligase-catalyzed reaction to close the final phosphodiester bond in newly synthesized DNA. ATP is required as a source of energy for this endergonic reaction.

16. DNA replication proteins

17. Eukaryotic DNA polymerases Several eukaryotic DNA polymerases also known:  found only in nucleus, requires template and primer.  also found in nucleus  found in mitochondria

18. Table 6–1 Error Rates in DNA synthesis If an incorrect nucleotide is added to a growing strand, the DNA polymerase will cleave it from the strand and replace it with the correct nucleotide before continuing.

19. DNA Pol I is a proofreading enzyme 3 active sites on the same enzyme: DNA polymerase activity, a 3’  5’ exonuclease and a 5’  3’ exonuclease. 5’  3’ exonuclease activity is independent of the 3’  5’ exonuclease and polymerase activity. Pol I can be cleaved by trypsin into two fragments: larger C- terminal fragment is called the Klenow fragment (KF) and contains the polymerase and 3’  5’ exonuclease activity. The N terminal fragment contains the 5’  3’ exonuclease activity. Pol I is a processive enzyme; it catalyzes a series of successive polymerization events (20 or more) without releasing the template.

20. Figure 11.7a Exonuclease activity of DNA polymerase I (a) 3’  5’ exonuclease activity DNA polymerase I acts as a proofreading and repair enzyme by catalyzing hydrolytic removal of mismatched bases.

21. Figure 11.7b Exonuclease activity of DNA polymerase I (b) 5’  3’ exonuclease activity DNA polymerase I acts as a proofreading and repair enzyme by catalyzing hydrolytic removal of mismatched bases.

22. Figure 30-10Schematic diagram for the nucleotidyl transferase mechanism of DNA polymerases. Page 1143 2 metal ions (Mg 2+ ) B ligands the phosphates of dNTP A bridges Pa with the primer’s 3’OH for in-line nucleophilic attack.

23. Pol I 3’-5’ exonuclease functions in editing DNA. Crystal structure shows a 12 nt “template” strand (5’- TGCCTCGCGGCC-3’) and a 7 nt “primer” strand (3’- GCGCCGG-5’) that is complementary to the 3’ end of the template strand and a 2’,3’-epoxy-ATP. Forms distorted segment. O 2’,3’-epoxy-ATP

24. Page 1141 template primer

25. Pol I A second primer strand is also present that base pairs with the 5’-terminal nucleotides but has a single T overhang on the template strand and a 3 nucleotide overhang on the primer strand. The 3’-terminal nucleotide of the primer strand is bound at the 3’-5’ exonuclease site (editing complex). The 3’ end of the primer strand is in competition to go between the active site and editing site. If properly base paired it goes to the active site, otherwise it goes in the editing site.

26. Figure 30-11Probable sequence of the double- stranded DNA seen in the X-ray structure of KF. Page 1144

27. Other Pol I activities Pol I functions to repair DNA by simultaneously filling in single-strand gaps formed during endonucleolytic cleavage of DNA. The 5’-3’ exonuclease and polyerase activities can replace nt on the 5’ side of a single strand nick. These reactions move the nick toward the 3’ end of the DNA strand (nick translation). Can be used to create radiolabeled DNA.

28. Page 1144 Figure 30-12Nick translation as catalyzed by Pol I.

29. Other Pol I activities The 5’-3’ exonuclease activity also removes RNA primers at the 5’ ends of newly synthesized DNA. DNA polymerase fills in gaps. Used temperature-sensitive E. coli mutants to demonstrate the function. Essential for survival.

30. DNA Pol III is the replicase Pol III core has multiple subunit composition. The polC gene encodes the polymerase function. Has a 3’  5’ exonuclease editing function. 5’  3’ exonuclease activity can only act on single stranded DNA. Pol III core has processivity for only 10-15 residues. Needs the  -subunit in the presence of the  complex for maximum processivity (>5000 residues).  -subunit forms a sliding clamp on the DNA.

31. Table 30-2Components of E. coli DNA Polymerase III Holoenzyme. Page 1145

32. Figure 30-13aX-Ray structure of the  subunit of E. coli Pol III holoenzyme. Ribbon drawing. Page 1146

33. Figure 30-13bThe  subunit of E. coli Pol III holoenzyme. Space-filling model of sliding clamp in hypothetical complex with B-DNA. Page 1146

34. DNA is unwound by Helicases Helicases are involved in DNA replication, recombination, repair and transcription termination, RNA splicing, and RNA editing. Translocate on one strand of double-helical nucleic acid to unwind the double helix using the energy of hydrolysis of NTPs. Classified by their direction: 5’-3’ and 3’-5’ helicases.

35. Table 30-3Unwinding and Binding Proteins of E. coli DNA Replication. Page 1146

36. Helicases DnaB-hexameric helicase, 5’-3’ translocation along the lagging strand, hydrolyzes ATP. Cannot use UTP. No crystal structure. T7 gene 4 helicase/primase-2-tiered hexagonal ring with N- terminal domains containing the primase activity and C-termini carry out the helicase activity. Uses dTTP (also dATP and ADP). Crystal structure known (Hexameric C-termini similar to DnaB).

37. Figure 30-14Unwinding of DNA by the combined action of DnaB and SSB proteins. Page 1147

38. Figure 30-15Electron microscopy–based image reconstruction of T7 gene 4 helicase/primase. Page 1147

39. Figure 30-16X-Ray structure of the helicase domain of T7 gene 4 helicase/primase. Page 1147

40. Helicases Rep helicase- monomeric enzyme, but active form is a dimer with 3’-5’ translocation. Shows negative cooperativity, one subunit binds to ssDNA and inhibits the binding of the other subunit to ssDNA(binds to dsDNA). Enzyme operates through “active/rolling” mechanism. Unwinds DNA by walking up strand in ATP dependent manner. PriA -is a similar monomeric enzyme with 3’-5’ translocation

41. Figure 30-17Active, rolling mechanism for DNA unwinding by Rep helicase. Page 1148

42. Figure 30-18X-Ray structure of Rep helicase in complex with dT(pT) 15 and ADP. Page 1148 ADP ssDNA Closed Open Two domains (1 and 2) Each domain is in 2 subdomains (1A, 2A-N-terminal subdomain) Change occurs in domain 2B from open to closed

43. Figure 30-19X-Ray structure of the N-terminal 135 residues of E. coli SSB in complex with dC(pC) 34. Page 1149 SSB prevent ssDNA from reannealing to form dsDNA Homotetramer 2 major forms (subscript is # of nt) (SSB) 35 in which 2 of of the four subunits interact with ssDNA (SSB) 35 shows unlimited cooperativity (SSB) 65 in which all 4 of the subunits interact with ssDNA; limited cooperativity. ssDNA

44. Figure 30-20The reactions catalyzed by E. coli DNA ligase. Page 1150 Can use either NAD + converted to NMN + +AMP or ATP to PP i and AMP. 1.The adenylyl group of NAD+ is transferred to  -amino group of Lys to form phosphoamide addict (unusual). 2.Adenylyl group of activated enzyme is transferred to 5’-phosphoryl terminus of nick to form adenylylated DNA (AMP linked to 5’nucleotide via PP i. 3.DNA ligase catalyzes formation of phosphodiester bond by attack of the 3’-OH group on the 5’-nucleotide.

45. Primase Closely associated with DNA helicase (T7 gene 4 helicase/primasehas both domains) E. coli primase (DnaG) forms noncovalent complex with DnaB Primase reverses its direction in order to synthesize RNA primer in 5’-3’ direction. 3 domains: N-terminal Zn 2+ binding domain, central catalytic domain with Mg 2+, C-terminal domain interacts with DnaB.

46. Figure 30-22X-Ray structure of E. coli primase. Page 1151