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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 7Ted YoungJanuary 19, 2011 Topics for today: DNA replication: forks, eyes, and DNA polymerase.
5. 5 Watson and Cricks 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.
8. 8
9. 9
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 Okazakis 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