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SCAN0010 - Protocol used by Campbell& Kleckner l t=0...

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Unformatted text preview: Protocol used by Campbell & Kleckner: l. t=0, start replication 42 C to 30 C using dnaCts cells 2. Isolate oriC plasmid at different t, restriction digest, 32P-label, assess binding to membrane proteins using nitrocellulose filter For t< 0.5 min, oriC DNA fragment doesn’t bind For 0.5 < t < ~12 min, some oriC fragment binds For t> 12 min, no oriC DNA fragment binds Conclude: When oriC region hemi—methylated, [email protected] to membrane protein, prevents initiation MODEL ° Hemimethylation of GATC’s mark oriC newly replicated - Prevents premature re—initiation - Binds to membrane protein, segregates 2 genomes? - Marks for mismatch repair 15 ...
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