Lecture14_DNAenz_2012 (1)

Lecture14_DNAenz_2012 (1) - 1 The search for truth is more...

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2 “The search for truth is more important than its Possession” Albert Einstein “Arts and science are not cast into a mould, but are formed and perfected by degrees, by often handling and polishing, as bears leisurely lick their cubs into form.” Michel de Montaigne
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Biochemistry 441 Lecture 14 Ted Young February 13, 2012 Biochemistry 441 Lecture 14 Ted Young February 13, 2012 Topic: DNA replication enzymes DNA polI pdb 3BDP β -clamp 2POL Watch this video for animation of the process: http:/wwwyoutube.com/watch?v=49fmm2WoWBs http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2614274 / Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III -subunit α Richard A. Wing, 1* Scott Bailey, 1,3* and Thomas A. Steitz J Mol Biol. 2008 October 17; 382(4): 859–869. Watch the short animation in the supplementary movie
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4 Polymerization-fidelity of polymerase Polymerization-fidelity of polymerase Normal bp fit into the polymerease active site but incorrect ones don’t. A-T, G-C are accommodated sterically; A-G, A-C, and G-T don’t fit as well. This contributes to the fidelity of replication. Base-pairing is less important than sterochemical “fit” (how would you test my assertion? Think synthetic organic chemistry-what kind of substituted bases would you make and why?) However, bases in the less-frequent tautomeric configuration “fit” normally and can be inserted so that for example, T would be incorporated opposite of G. This leads to a mutation if not repaired (more on this later). In vitro replication infidelity (insertion of an incorrect nucleotide) is estimated to be about 1 incorrectly inserted base per 10e4-5 bases whereas in vivo it is about 1 per 10e9-10. These errors are corrected by the editing function of the polymerase as well as post-replication repair pathways.
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5 Finding genes/proteins involved in DNA replication Finding genes/proteins involved in DNA replication Genetic approach- 1. isolate temperature- sensitive (ts) mutants. 2. screen for DNA synthesis at the restrictive temperature 3. characterize mutants defective in DNA synthesis 4. Identify biochemical defect Biochemical approach 1. Devise an assay (what function are you looking for?) 2. Break open cells and apply assay 3. Demonstrate that the protein identified plays an important role in vivo.
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6 Six enzymes to know-substrates, products, co- factors, and function in DNA metabolism Six enzymes to know-substrates, products, co- factors, and function in DNA metabolism DNA polymerase β -clamp/processivity factor DNA ligase DNA helicase Single-strand binding protein Telomerase These are enzymes/proteins that consist of more than one polypeptide chain; altogether about 160 genes are required for replication in all eukaryotes, from yeast to humans. Why so many?
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Lecture14_DNAenz_2012 (1) - 1 The search for truth is more...

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