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

  20. Helicase • Operates in replication fork • Separates strands to allow DNA Pol to function on single strands. • Involves breaking H-bonds and hydrophobic interactions • Requires ATP

  21. Initiation of Replication • Replicaion 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. 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

  29. 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

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