1. REPLICATION Chapter 7

2. The Problem DNA is maintained in a compressed, supercoiled state. DNA is maintained in a compressed, supercoiled state. BUT, basis of replication is the formation of strands based on specific bases pairing with their complementary bases. BUT, basis of replication is the formation of strands based on specific bases pairing with their complementary bases.  Before DNA can be replicated it must be made accessible, i.e., it must be unwound  Before DNA can be replicated it must be made accessible, i.e., it must be unwound

3. THREE HYPOTHESES FOR DNA REPLICATION Models of Replication

4. (a) Hypothesis 1: Semi-conservative replication (b) Hypothesis 2: Conservative replication Intermediate molecule (c) Hypothesis 3: Dispersive replication MODELS OF DNA REPLICATION

5. PREDICTED DENSITIES OF NEWLY REPLICATED DNA MOLECULES ACCORDING TO THE THREE HYPOTHESES ABOUT DNA REPLICATION

6. Meselson and Stahl Conclusion: Semi-conservative replication of DNA

7. Replication as a process Double-stranded DNA unwinds. Double-stranded DNA unwinds. The junction of the unwound molecules is a replication fork. A new strand is formed by pairing complementary bases with the old strand. Two molecules are made. Each has one new and one old DNA strand.

8. Extending the Chain dNTPs are added individually dNTPs are added individually Sequence determined by pairing with template strand Sequence determined by pairing with template strand DNA has only one phosphate between bases, so why use dNTPs? DNA has only one phosphate between bases, so why use dNTPs?

9. Extending the Chain Extending the Chain

10. DNA Synthesis 2 phosphates 3’-OH nucleophilic attack on alpha phosphate of incoming dNTP removal and splitting of pyrophosphate by inorganic pyrophosphatase

11. Chain Elongation in the 5’  3’ direction

12. Semi-discontinuous Replication All known DNA pols work in a 5’>>3’ direction All known DNA pols work in a 5’>>3’ direction Solution? Solution? Okazaki fragments Okazaki fragments

13. Okazaki Experiment

14. Continuous synthesis Discontinuous synthesis  DNA replication is semi-discontinuous

15. Features of DNA Replication DNA replication is semiconservative DNA replication is semiconservative Each strand of template DNA is being copied. Each strand of template DNA is being copied. DNA replication is semidiscontinuous DNA replication is semidiscontinuous The leading strand copies continuously The leading strand copies continuously The lagging strand copies in segments (Okazaki fragments) which must be joined The lagging strand copies in segments (Okazaki fragments) which must be joined DNA replication is bidirectional DNA replication is bidirectional Bidirectional replication involves two replication forks, which move in opposite directions Bidirectional replication involves two replication forks, which move in opposite directions

16. DNA Replication-Prokaryotes DNA replication is semiconservative.  the helix must be unwound. DNA replication is semiconservative.  the helix must be unwound. Most naturally occurring DNA is slightly negatively supercoiled. Most naturally occurring DNA is slightly negatively supercoiled.  Torsional strain must be released  Torsional strain must be released Replication induces positive supercoiling Replication induces positive supercoiling  Torsional strain must be released, again.  Torsional strain must be released, again. SOLUTION: Topoisomerases SOLUTION: Topoisomerases

17. The Problem of Overwinding

18. Topoisomerase Type I Precedes replicating DNA Precedes replicating DNA Mechanism Mechanism Makes a cut in one strand, passes other strand through it. Seals gap. Makes a cut in one strand, passes other strand through it. Seals gap. Result: induces positive supercoiling as strands are separated, allowing replication machinery to proceed. Result: induces positive supercoiling as strands are separated, allowing replication machinery to proceed.

19. Helicase Operates in replication fork Operates in replication fork Separates strands to allow DNA Pol to function on single strands. Separates strands to allow DNA Pol to function on single strands. Translocate along single strain in 5’->3’ or 3’-> 5’ direction by hydrolyzing ATP

20. Gyrase--A Type II Topoisomerase Introduces negative supercoils Introduces negative supercoils Cuts both strands Cuts both strands Section located away from actual cut is then passed through cut site. Section located away from actual cut is then passed through cut site.

21. Initiation of Replication Replication initiated at specific sites: Origin of Replication (ori) Replication initiated at specific sites: Origin of Replication (ori) Two Types of initiation: Two Types of initiation: De novo –Synthesis initiated with RNA primers. Most common. De novo –Synthesis initiated with RNA primers. Most common. Covalent extension—synthesis of new strand as an extension of an old strand (“Rolling Circle”) Covalent extension—synthesis of new strand as an extension of an old strand (“Rolling Circle”)

22. De novo Initiation Binding to Ori C by DnaA protein Binding to Ori C by DnaA protein Opens Strands Opens Strands Replication proceeds bidirectionally Replication proceeds bidirectionally

23. Unwinding the DNA by Helicase (DnaB protein) Uses ATP to separate the DNA strands Uses ATP to separate the DNA strands At least 4 helicases have been identified in E. coli. At least 4 helicases have been identified in E. coli. How was DnaB identified as the helicase necessary for replication? How was DnaB identified as the helicase necessary for replication? NOTE: Mutation in such an essential gene would be lethal. NOTE: Mutation in such an essential gene would be lethal. Solution? Solution? Conditional mutants Conditional mutants

24. Liebowitz Experiment What would you expect if the substrates are separated by electrophoresis after treatment with a helicase?

25. Liebowitz Assay--Results What do these results indicate? What do these results indicate? ALTHOUGH PRIMASE (DnaG) AND SINGLE- STRAND BINDING PROTEIN (SSB) BOTH STIMULATE DNA HELICASE (DnaB), NEITHER HAVE HELICASE ACTIVITY OF THEIR OWN ALTHOUGH PRIMASE (DnaG) AND SINGLE- STRAND BINDING PROTEIN (SSB) BOTH STIMULATE DNA HELICASE (DnaB), NEITHER HAVE HELICASE ACTIVITY OF THEIR OWN

26. Single Stranded DNA Binding Proteins (SSB) Maintain strand separation once helicase separates strands Maintain strand separation once helicase separates strands Not only separate and protect ssDNA, also stimulates binding by DNA pol (too much SSB inhibits DNA synthesis) Not only separate and protect ssDNA, also stimulates binding by DNA pol (too much SSB inhibits DNA synthesis) Strand growth proceeds 5’>>3’ Strand growth proceeds 5’>>3’

27. Replication: The Overview Requirements: Requirements: Deoxyribonucleotides Deoxyribonucleotides DNA template DNA template DNA Polymerase DNA Polymerase 5 DNA pols in E. coli 5 DNA pols in E. coli 5 DNA pols in mammals 5 DNA pols in mammals Primer Primer Proofreading Proofreading

28. A total of 5 different DNAPs have been reported in E. coli DNAP I: functions in repair and replication DNAP II: functions in DNA repair (proven in 1999) DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999) DNAP V: functions in DNA repair (discovered in 1999) To date, a total of 14 different DNA polymerases have been reported in eukaryotes The DNA Polymerase Family

30. DNA pol I First DNA pol discovered. First DNA pol discovered. Proteolysis yields 2 chains Proteolysis yields 2 chains Larger Chain (Klenow Fragment) 68 kd Larger Chain (Klenow Fragment) 68 kd C-terminal 2/3rd. 5’>>3’ polymerizing activity C-terminal 2/3rd. 5’>>3’ polymerizing activity N-terminal 1/3rd. 3’>>5’ exonuclease activity N-terminal 1/3rd. 3’>>5’ exonuclease activity Smaller chain: 5’>>3 exonucleolytic activity Smaller chain: 5’>>3 exonucleolytic activity nt removal 5’>>3’ nt removal 5’>>3’ Can remove >1 nt Can remove >1 nt Can remove deoxyribos or ribos Can remove deoxyribos or ribos

31. DNA pol I First DNA pol discovered. First DNA pol discovered. Proteolysis yields 2 chains Proteolysis yields 2 chains Larger Chain (Klenow Fragment) 68 kd Larger Chain (Klenow Fragment) 68 kd C-terminal 2/3rd. 5’>>3’ polymerizing activity C-terminal 2/3rd. 5’>>3’ polymerizing activity N-terminal 1/3rd. 3’>>5’ exonuclease activity N-terminal 1/3rd. 3’>>5’ exonuclease activity Smaller chain: 5’>>3 exonucleolytic activity Smaller chain: 5’>>3 exonucleolytic activity nt removal 5’>>3’ nt removal 5’>>3’ Can remove >1 nt Can remove >1 nt Can remove deoxyribos or ribos Can remove deoxyribos or ribos

32. The structure of the Klenow fragment of DNAP I from E. coli

33. Requires 5’-3’ activity of DNA pol I Steps 1.At a nick (free 3’ OH) in the DNA the DNA pol I binds and digests nucleotides in a 5’-3’ direction 2.The DNA polymerase activity synthesizes a new DNA strand 3.A nick remains as the DNA pol I dissociates from the ds DNA. 4.The nick is closed via DNA ligase Nick Translation Source: Lehninger pg. 940

34. 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation" 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation" This activity is critical in primer removal Nick Translation 2

35. DNA Polymerase I is great, but…. In 1969 John Cairns and Paula deLucia -isolated a mutant bacterial strain with only 1% DNAP I activity (polA) - mutant was super sensitive to UV radiation - but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA Conclusion: DNA pol I is NOT the principal replication enzyme in E. coli DNA pol I is NOT the principal replication enzyme in E. coli

36. - DNAP I is too slow (600 dNTPs added/minute) - DNAP I is only moderately processive (processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template) Conclusion: There must be additional DNA polymerases. There must be additional DNA polymerases. Biochemists purified them from the polA mutant Biochemists purified them from the polA mutant Other clues….

37. The major replicative polymerase in E. coli ~ 1,000 dNTPs added/sec It’s highly processive: >500,000 dNTPs added before dissociating Accuracy: 1 error in 10 7 dNTPs added, with proofreading final error rate of 1 in 10 10 overall. DNA Polymerase III

38. The 10 subunits of E. coli DNA polymerase III SubunitFunction ’’ 5’ to 3’ polymerizing activity 3’ to 5’ exonuclease activity  and  assembly (scaffold) Assembly of holoenzyme on DNA Sliding clamp = processivity factor Clamp-loading complex Core enzyme Holoenzyme DNA Polymerase III Holoenzyme (Replicase)

39. Activities of DNA Pol III ~900 kd Synthesizes both leading and lagging strand Can only extend from a primer (either RNA or DNA), not initiate 5’>>3’ polymerizing activity 3’>>5’ exonuclease activity NO 5’>>3’ exonuclease activity

40. Subsequent hydrolysis of PPi drives the reaction forward Nucleotides are added at the 3'-end of the strand The 5’ to 3’ DNA polymerizing activity

41. Leading and Lagging Strands REMEMBER: DNA polymerases require a primer. REMEMBER: DNA polymerases require a primer. Most living things use an RNA primer Most living things use an RNA primer Leading strand (continuous): primer made by RNA polymerase Leading strand (continuous): primer made by RNA polymerase Lagging strand (discontinuous): Primer made by Primase Lagging strand (discontinuous): Primer made by Primase Priming occurs near replication fork,  need to unwind helix. SOLUTION: Helicase Priming occurs near replication fork,  need to unwind helix. SOLUTION: Helicase Primosome= Primase + Helicase Primosome= Primase + Helicase

42. The Replisome DNA pol III extends on both the leading and lagging strand DNA pol III extends on both the leading and lagging strand Growth stops when Pol III encounters an RNA primer (no 5’>>3’ exonuclease activity) Growth stops when Pol III encounters an RNA primer (no 5’>>3’ exonuclease activity) Pol I then extends the chain while removing the primer (5’>>3’) Pol I then extends the chain while removing the primer (5’>>3’) Stops when nick is sealed by ligase Stops when nick is sealed by ligase

43. Ligase Uses NAD + or ATP for coupled reaction Uses NAD + or ATP for coupled reaction 3-step reaction: 3-step reaction: AMP is transferred to Lysine residue on enzyme AMP is transferred to Lysine residue on enzyme AMP transferred to open 5’ phosphate via temporary pyrophosphate (i.e., activation of the phosphate in the nick) AMP transferred to open 5’ phosphate via temporary pyrophosphate (i.e., activation of the phosphate in the nick) AMP released, phosphodiester linkage made AMP released, phosphodiester linkage made NAD  NMN + AMP NAD  NMN + AMP ATP  ADP + PPi ATP  ADP + PPi

44. DNA Replication Model 1. Relaxation of supercoiled DNA. 2. Denaturation and untwisting of the double helix. 3. Stabilization of the ssDNA in the replication fork by SSBs. 4. Initiation of new DNA strands. 5. Elongation of the new DNA strands. 6. Joining of the Okazaki fragments on the lagging strand.

45. Termination of Replication Occurs @ specific site opposite ori c Occurs @ specific site opposite ori c ~350 kb ~350 kb Flanked by 6 nearly identical non-palindromic*, 23 bp terminator (ter) sites Flanked by 6 nearly identical non-palindromic*, 23 bp terminator (ter) sites * Significance? * Significance? Tus Protein-arrests replication fork motion

46. FIDELITY OF REPLICATION Expect 1/10 3-4, get 1/10 8-10. Expect 1/10 3-4, get 1/10 8-10. Factors Factors 3’  5’ exonuclease activity in DNA pols 3’  5’ exonuclease activity in DNA pols Use of “tagged” primers to initiate synthesis Use of “tagged” primers to initiate synthesis Battery of repair enzymes Battery of repair enzymes Cells maintain balanced levels of dNTPs Cells maintain balanced levels of dNTPs

47. Why Okazaki Frags? Or, why not 3’  5’ synthesis? Or, why not 3’  5’ synthesis? Possibly due to problems with proofreading. Possibly due to problems with proofreading. PROBLEM: PROBLEM: Imagine a misincorporation with a 3’  5’ polymerase Imagine a misincorporation with a 3’  5’ polymerase How is it removed? How is it removed? How is the chain extended? How is the chain extended? How is the chain extended? How is the chain extended? Is there a problem after removing a mismatch? Is there a problem after removing a mismatch?

48. Covalent Extension Methods Often called “Rolling circle” Often called “Rolling circle” Common in bacteriophages Common in bacteriophages NOTE: de novo initiation of circular DNA results in theta structures, sometimes callled “theta replication” NOTE: de novo initiation of circular DNA results in theta structures, sometimes callled “theta replication”

49. Rolling Circle I Few rounds of theta- replication Few rounds of theta- replication Nick outer strand Nick outer strand Extend 3’ end of outer strand, displacing original Extend 3’ end of outer strand, displacing original Synthesis of complementary strand using displaced strand as template Synthesis of complementary strand using displaced strand as template Concatamers cut by RE’s, sealed Concatamers cut by RE’s, sealed Result several copies of circular dsDNA Result several copies of circular dsDNA

50. Rolling Circle I “Template “rolls”, extrudes leading strand “Template “rolls”, extrudes leading strand Okazaki frags made on leading strand as it emerges. Okazaki frags made on leading strand as it emerges.

51. Rolling Circle I NOTE: can get single unit genomes or multimeric copies

52. Rolling Circle II EX ΦX174 EX ΦX174 Circular ssDNA chromosome Circular ssDNA chromosome Copy + strand using E. coli replication proteins to make ds circle (theta replication) Copy + strand using E. coli replication proteins to make ds circle (theta replication) Protein A (phage) cuts + strand Protein A (phage) cuts + strand Rolling circle replication Rolling circle replication Protein A cuts at unit length and circularizes (ligates) released ss chromosome Protein A cuts at unit length and circularizes (ligates) released ss chromosome Replication continues Replication continues

53. Reverse Transcription DNA replication in retroviruses DNA replication in retroviruses RNA Dependent DNA polymerase RNA Dependent DNA polymerase Process: Process: Retroviral RNA acts as template Retroviral RNA acts as template Primer—Segment of host cell t-RNA Primer—Segment of host cell t-RNA Result: DNA RNA hybrid Result: DNA RNA hybrid RNA strand degraded by RNAse H RNA strand degraded by RNAse H DNA strand serves as template. DNA strand serves as template. Also catalyzed by RT Also catalyzed by RT Result:dsDNA Result:dsDNA New DNA integrates into host genome New DNA integrates into host genome

54. cDNA Library Made from mRNA Made from mRNA Steps Steps 1 st strand 1 st strand RNAse H RNAse H 2 nd strand 2 nd strand Tailing Tailing Insertion Insertion Transform Transform

55. Eukaryotic DNA Replication Much larger genomes with slower polymerase Much larger genomes with slower polymerase Solution Solution Multiple initiation sites Multiple initiation sites More molecules of polymerase More molecules of polymerase EX: DNA pol  present in ~2-5 X10 5 copies/cell EX: DNA pol  present in ~2-5 X10 5 copies/cell Histones an issue Histones an issue Still many questions Still many questions

56. Completing the Ends of Non-circular DNA THE PROBLEM? THE PROBLEM? Solutions Solutions Phage T-7 Phage T-7 Eukaryotes Eukaryotes

57. Phage Solution to Problem Phage DNA is linear Phage DNA is linear Ends have repetitive complementary sequences Ends have repetitive complementary sequences After removal of 5’end RNA primer, are left with a 3’ overhang After removal of 5’end RNA primer, are left with a 3’ overhang Overhangs form H-bonds with complementary overhangs, gaps filled in ligated. RESULT: concatamers Overhangs form H-bonds with complementary overhangs, gaps filled in ligated. RESULT: concatamers RE cuts concatamer into unit length genomes with 5’ overhangs RE cuts concatamer into unit length genomes with 5’ overhangs DNA pol extends 3’ ends, resulting in complete unit length genomes. DNA pol extends 3’ ends, resulting in complete unit length genomes.

58. Eukaryotes Solution: Telomeres Solution: Telomeres At ends of chromosomesare non-coding regions, >1000 tandem repeats of GC rich sequence. At ends of chromosomesare non-coding regions, >1000 tandem repeats of GC rich sequence. Telomeric DNA synthesized and maintained by Telomerase Telomeric DNA synthesized and maintained by Telomerase Adds tandem repeats of TTGGG Adds tandem repeats of TTGGG Is a ribonucleoprotein, uses internal ribonucleotide sequences as a template Is a ribonucleoprotein, uses internal ribonucleotide sequences as a template

59. Telomeres Elongation Elongation Translocation Translocation Elongation Elongation New primer synthesis New primer synthesis Dna Replication Dna Replication Primer removal Primer removal Repeat Repeat