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Lecture 7

BIO372 - Lecture 7.pdf

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University of Toronto Mississauga
Gordon Anderson

Lecture 7 January-30-14 10:13 AM Helicases unwind DNA Many enzymes/ protein are ATP dependent - capability is widespread Helicases is one of them DNA-replication-deficientmutants in E. coli We want conditional mutants - 30 degrees is nice temp for E coli to grow - We don’t want 40 degrees Type 2 has some growth at 40 but plataeus after Type 1 is deficient in helicase (at 40 degrees, E. coli didn’t grow right away) Helicase assay DnaB - impt in helicase, has an ATPase activity Circular DNA single stranded - Some of the segmentswill have nicks We take probe DNA and label 5' end (what kind of label, substrate - need to know this) Substrate runs slowly - why two bands? Because of the nicks, not because of super coiling You can measure product to have an idea how helicase is working You have to have protein to get products Amount of products varies dnaB and SSB has an effect Relationship between SSB and dnaG - synergistic DNA Replication, Part 2 Single-strand binding proteins Table 20.1 - don't have to memorize Fewer copies per cell will have more activity Stimulation of DNA synthesis by T4 phage SSB, gp32 Look at short region of synthesis, and the rate of incorporation of labeled DNA If there's gp32, DNA replication goes up More of gp32, DNA keeps going up. But if there's too much, replication goes back down. The ratio must be optimal (enough substrates for enough enzymes) There's so much substrate that the active site becomesclogged = substrate inhibition How is this possible? Effect of the SSB gene 32 Production of DNA at 2 temp (42 and 25) There's little effect over time Each construct has a double mutant (not good, only change one thing at a time) Do all 8 combinations if you wanna do double mutant Do
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