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REPLICATION

REPLICATION. Chapter 7. The Problem. 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.  Before DNA can be replicated it must be made accessible, i.e., it must be unwound.

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REPLICATION

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  1. REPLICATION Chapter 7

  2. The Problem • 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. •  Before DNA can be replicated it must be made accessible, i.e., it must be unwound

  3. Models of Replication THREE HYPOTHESES FOR DNA REPLICATION

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

  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. 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 • Sequence determined by pairing with template strand • DNA has only one phosphate between bases, so why use dNTPs?

  9. Extending the Chain

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

  11. Chain Elongation in the5’  3’ direction

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

  13. Okazaki Experiment

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

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

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

  17. The Problem of Overwinding

  18. Topoisomerase Type I • Precedes replicating DNA • Mechanism • 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.

  19. Helicase • Operates in replication fork • 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 • Cuts both strands • 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) • Two Types of initiation: • De novo –Synthesis initiated with RNA primers. Most common. • 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 • Opens Strands • Replication proceeds bidirectionally

  23. Unwinding the DNA by Helicase (DnaB protein) • Uses ATP to separate the DNA strands • At least 4 helicases have been identified in E. coli. • How was DnaB identified as the helicase necessary for replication? • NOTE: Mutation in such an essential gene would be lethal. • Solution? • 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? • 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 • Not only separate and protect ssDNA, also stimulates binding by DNA pol (too much SSB inhibits DNA synthesis) • Strand growth proceeds 5’>>3’

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

  28. The DNA Polymerase Family 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

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

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

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

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

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

  34. 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

  35. Other clues…. - 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. • Biochemists purified them from the polA mutant

  36. DNA Polymerase III 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 107 dNTPs added, • with proofreading final error rate of 1 in 1010 overall.

  37. The 10 subunits of E. coli DNA polymerase III 5’ to 3’ polymerizing activity 3’ to 5’ exonuclease activity a and e assembly (scaffold) Assembly of holoenzyme on DNA Sliding clamp = processivity factor Clamp-loading complex Clamp-loading complex Clamp-loading complex Clamp-loading complex Clamp-loading complex a e q t b g d d’ c y Core enzyme Holoenzyme DNA Polymerase III Holoenzyme (Replicase) Subunit Function

  38. 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

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

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

  41. The Replisome • 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) • Pol I then extends the chain while removing the primer (5’>>3’) • Stops when nick is sealed by ligase

  42. Ligase • Uses NAD+ or ATP for coupled reaction • 3-step reaction: • 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 released, phosphodiester linkage made • NADNMN + AMP • ATP ADP + PPi

  43. DNA Replication Model • Relaxation of supercoiled DNA. • Denaturation and untwisting of the double helix. • Stabilization of the ssDNA in the replication fork by SSBs. • Initiation of new DNA strands. • Elongation of the new DNA strands. • Joining of the Okazaki fragments on the lagging strand.

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

  45. FIDELITY OF REPLICATION • Expect 1/103-4, get 1/108-10. • Factors • 3’5’ exonuclease activity in DNA pols • Use of “tagged” primers to initiate synthesis • Battery of repair enzymes • Cells maintain balanced levels of dNTPs

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

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

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

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

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