BCH311H1 Chapter Notes - Chapter 3: Proliferating Cell Nuclear Antigen, Thymidine Triphosphate, Dna Replication
3 Mar 2013
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Dna replication: thymidine with atp and mg with e. coli extract. Treated with dnase to be acid soluble: found thymidine kinase created real substrate (dttp, all 4 dntps needed, same at:gc ratio as primer dna. Dna fidelity: polmerase and 3" --> 5" exonuclease site distinct. Mismatch causes distortion, slowing and opportunity for 3" end of dna strand to move into exonuclease site. Shape discrimination (mismatches disturbs enzyme) 10-5 (rna pol) 10-7: pol iii is the polymerase for replication chromosome in vivo. Replication is semi-conservative: this experiment by meselson and stahl that you really should know perfectly by now. Replication is bi-directional (cairns: 3h-thymidine labelled cells lysed and dna spread on slide. Coated with photographic emulsion and wait for years: observed theta structures showing two replication forks, short labelling. Where thick red is from the initial pulse labelling. Some with no pulse in middle- already in process of replication before initial pulse.
