Ch 11 - Ch 11(pg 281-299 Semiconservative DNA Replication o...

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Ch 11 (pg 281-299) Semiconservative DNA Replication o At time of Watson & Crick (1953), 3 competing DNA replication theories: semiconservative, conservative, dispersivel o Meselson-Stahl Experiment (1958) separate 14 N from 15 N w/ centrifuging w/ CsCl density gradient grew E. coli in medium w/ only nitrogen source being 15 N all DNA had 15 N took 15 N-labled bacteria into medium w/ 14 N gen 0: all DNA at high density gen 1: intermediate DNA gen 2: ½ intermediate DNA, ½ at low density meant semi-conservative if conservative: gen 1 ½ high density, ½ low density if dispersive: gen 2 all DNA ½ btwn intermediate and low density o Semiconservative DNA Replication in Eukaryotes see by staining replicating chrom w/ 5-bromodeoxyuridine (BUdR) BUdR – base analog similar to T (incorporated instead of T) lighter as amt of BUdR incorporated increases Chinese hamster ovary cells treated w/ BUdR gen 1: all T strand—BUdR strand gen 2: 1 T strand—BudR strand:1 BUdR strand—BudR strand o harlequin chrom: mitotic chrom w/ 1 darkly stained sister DNA Polymerases, the DNA Replicating Enzymes o 1955 – Kornberg identified DNA replication enzymes – got Nobel Prize o DNA Pol I (aka Kornberg enzyme) mix radioactively-labeled dNTPs w/ E. coli lysate (cells of bacteria broken open to release contents) DNA synthesis isolated DNA pol I 4 components for DNA synthesis all 4 dNTPs DNA fragment as template DNA pol I Mg 2+ for optimal DNA pol activity o Roles of DNA Pols features of DNA pols at growing end of DNA chain, they catalyze formation of 4 of dNTP precursor o point: lengthening DNA chain acts as a primer – a preexisting polynucleotide to which a new nucleotide can be added to free 3’-OH w/ high accuracy, DNA pol finds correct precursor dNTP that can form a complementary bp w/ the nucleotide on the template
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5’ 3’ synthesis studied DNA replication enzymes by inducing a mutation in the gene that codes for the enzymes (after thinking only 1 enzyme in DNA replication in E. coli) 3’ exonuclease activity o mutation not lethal/crippling – so enzyme not essential to cell function o cells w/ polA1 mutation have high mutation rates – so DNA pol I has important function in repairing damaged DNA polAex1 o
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Ch 11 - Ch 11(pg 281-299 Semiconservative DNA Replication o...

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