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lecturenoteschap20-09-4slides - BCMB 3100 Chapter 20 DNA...

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1 BCMB 3100 - Chapter 20 DNA Replication Semi-conservative DNA replication DNA polymerase DNA replication Replication fork; Okazaki fragments Sanger method for DNA sequencing DNA repair PCR Fig 20.1 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 Enzymatic Synthesis of DNA Arthur Kornberg (1955-58) discovered an enzyme that synthesized DNA Experimental Strategy 1) dNTPs as precursors of DNA 2) sensitive assay to detect newly synthesized DNA; 3) When animal cell extracts proved unsuccesful they turned to E. coli E. coli divides fast (every 20 minutes) and large quantities of cells can be isolated Results of Kornberg experiments (1955-58) E. coli extract + 14 C-labeled dTTP (1,000,000 cpm) incubate acid precipitate dTTP dTTP ~ 1,000,000 cpm 50 cpm First evidence for DNA polymerase! 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 100 kg (~220 lbs) E. coli 500 mg DNA Polymerase I
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2 DNA Polymerase I Molecular Weight: 103 kd; monomer Activity (DNA) n residues + dNTP (DNA) n+1 + PPi 2 Pi Requirements: dATP, dTTP, dGTP, dCTP Mg++ Primer with free 3’-OH Template (single stranded DNA) 5’ _________________ OH 3’ + dATP + DNA polymerase 3’__________________________T_____________5’ 5’ ______________________A OH 3 ’+ PP i 3’_______________________T_____________5’ primer template DNA Polymerase is template-directed ** one active site (for polymerase activity ) can accommodate all four dNTPs; the correct dNTP is determined by the corresponding base on the template stand. DNA Polymerase I is moderately processive (~20 residues) *Polymerization is in the 5’ 3’ direction E. coli DNA Polymerase I has three different active sites on a single polypeptide chain!! Activities of DNA Polymerase I 1) 5’ 3’ polymerase 2) 3’ 5’ exonuclease (proof-reading) 3) 5’ 3’ exonuclease (editing) Proof-reading: 3’ 5’ Exonuclease Activity 5’ …pTpApGpCp C -OH 3’ …pApTpCpGp A pTpCpGpApT…5’ 3’-5’ Exonuclease activity A-C mismatch 5’ …pTpApGpCp T -OH 3’ …pApTpCpGp A pTpCpGpApT…5’ Correct base can then be inserted
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lecturenoteschap20-09-4slides - BCMB 3100 Chapter 20 DNA...

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