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It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mech

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It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mech

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    3. 3 “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” J.D. Watson F.H.C. Crick Nature (1953) p 737.

    4. 4 Biochemistry 441 Lecture 7 Ted Young January 19, 2011 Topics for today: DNA replication: forks, eyes, and DNA polymerase.

    5. 5 Watson and Crick’s proposal Proof of semi-conservative replication: the Meselson-Stahl density transfer experiment.

    6. 6 Observations of DNA replicating in vivo Conclusions from experiments using tritium autoradiography: 1. One double-stranded DNA molecule/chromosome for all prokaryotes and eukaryotes. 2. One replication origin per chromosome (bacteria/viruses). Multiple origins/chromosome for eukaryotes

    7. 7 Observations of DNA replicating in vivo Tritium autoradiography: 3. Bidirectional replication. 4. Both strands replicate simultaneously.

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    10. 10 A paradox? The observation that both template strands were copied at each replication fork was difficult to reconcile with the mode of 5’- to 3’ elongation. Semi-discontinuous replication resolved the paradox.

    11. 11 Confirmation of discontinuous replication model 1. Pulse-labeled DNA was present in short fragments (Okazaki fragments). 2. Single-stranded DNA was observed at each replication fork.

    12. 12 Getting started in DNA replication-the role of RNA primers Okazaki’s second discovery-DNA replication is initiated using an RNA primer-32P-ribonucleotide transfer experiment.

    13. 13 DNA replication enzymology- DNA polymerase Properties of DNA polymerase enzymes: many have more than one active site 1. Polymerization site-all enzymes 2. 3’-5’ exonuclease “editing” site-most enzymes 3. 5’-3’ exonuclease “nick-translation” site-a few enzymes

    14. 14 5’- to 3’-polymerization of dNTPs by DNA polymerase Polynucleotide chain growth is always anti-parallel. All DNA and RNA polymerases use the same basic mechanism.

    15. 15 Nucleotide polymerization Note direction of chain growth: 5’ to 3’

    16. 16 Deoxyribonucleotide polymerization

    17. 17 Most DNA polymerases have other activities as well….

    18. 18 DNA polymerases can have other activities as well…. 3’-5’-exonuclease “editing” or “proofreading” activity-most DNA polymerases. 5’-3’ exonuclease “nick-translating” activity-some bacterial DNA polymerases

    19. 19 Model of 3’-5’ exonuclease “editing” by DNA polI

    20. 20 3’-5’ exonuclease “editing” by DNA polI

    21. 21 5’-3’ exonuclease activity-”nick-translation”

    22. 22 X-ray structure of poI Klenow fragment with DNA The Klenow fragment is the C-terminal 2/3 of polI and contains two separate catalytic domains: a polymerization and a 3’-5’ exonuclease domain. The 5’ exo domain is in the N-terminus of the enzyme.

    23. 23 Another model for polymerization and editing

    24. 24 DNA replication summary In vivo DNA is replicated… semiconservatively, starting at unique sites (origins), semi-discontinuously, using RNA primers. In vitro numerous DNA polymerases have been characterized. They all polymerize DNA 5’ to 3’ using a single-stranded DNA template, a primer, and 5’-dNTP. Most have associated nuclease activities, either as part of the same polypeptide chain, or on another subunit of of multi-subunit enzyme

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