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lecturenoteschap20-10-1slide - BCMB 3100 - Chapter 20 DNA...

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BCMB 3100 - Chapter 20 DNA Replication Semi-conservative DNA replication DNA polymerase po y e ase DNA replication Replication fork; Okazaki fragments Sanger method for DNA sequencing DNA repair PCR
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Fig 20.1 Meselson & Stahl, 1958 Semiconservative DNA replication Each strand of DNA acts as a template for synthesis of a new strand Daughter DNA contains one parental and one newly synthesized strand
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Enzymatic Synthesis of DNA Arthur Kornberg (1955-58) discovered an enzyme at synthesized DNA that synthesized DNA Experimental Strategy 1) dNTPs as precursors of DNA 2) sensitive assay to detect newly synthesized DNA; radioactive dNTPs & acid precipitation of DNA 3) When animal cell extracts proved unsuccesful they turned to E. coli co l i ivides fast (every 20 minutes) and large E. coli divides fast (every 20 minutes) and large quantities of cells can be isolated
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Results of Kornberg experiments (1955-58) E. coli extract + 14 C-labeled dTTP (1,000,000 cpm) cubate incubate acid precipitate dTTP dTTP ~ 1,000,000 cpm 50 cpm First evidence for DNA polymerase! 0 / 1,000,000 cpm .005% of radioactivity incorporated into DNA 50 / 1,000,000 cpm 0.005% of radioactivity incorporated into DNA Enzyme purification DNA Polymerase I Took approximately 10 years to purify and characterize 00 kg (~220 lbs) E coli 00 mg DNA Polymerase I 100 kg (~220 lbs) E. coli 500 mg DNA Polymerase I
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NA Polymerase I DNA Polymerase I Molecular Weight: 103 kd; monomer ctivity Activity (DNA) n residues + dNTP (DNA) n+1 + PPi 2 Pi Requirements: dATP, dTTP, dGTP, dCTP Mg++ rimer with free 3’ H Primer with free 3 -OH Template (single stranded DNA) H dATP + DNA polymerase primer 5 _________________ OH 3 + dATP + DNA polymerase 3’__________________________T_____________5’ template 5’ ______________________A OH 3 + PPi 3’_______________________T_____________5’
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NA Polymerase is template- irected DNA Polymerase is template directed ** one active site (for polymerase activity ) can ccommodate all four dNTPs; the correct accommodate all four dNTPs; the correct dNTP is determined by the corresponding ase on the template stand. base on the template stand. DNA Polymerase I is moderately processive (~20 residues) * olymerization is in the 5’ ’ direction Polymerization is in the 5 3 direction
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E. coli DNA Polymerase I has three different active sites on a single polypeptide chain!! ctivities of DNA Polymerase I Activities of DNA Polymerase I 1) 5’ 3’ polymerase 2) 3’ 5’ exonuclease (proof-reading) 3) 5’ 3’ exonuclease (editing)
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Proof-reading: 3’ 5’ Exonuclease Activity 3’-5’ Exonuclease activity 5’ …pTpApGpCp C -OH 3’ …pApTpCpGp A pTpCpGpApT…5’ A-C mismatch 5’ …pTpApGpCp T -OH 3’ …pApTpCpGp A pTpCpGpApT…5’ Correct base can then be inserted *DNA polymerase I examines the result of each polymerization before proceeding to the next.
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Editing: 5’ 3’ Exonuclease Activity
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This note was uploaded on 12/03/2011 for the course CHEM 3100 taught by Professor Dervartanian during the Fall '09 term at University of Georgia Athens.

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lecturenoteschap20-10-1slide - BCMB 3100 - Chapter 20 DNA...

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