412U3 - Replication: general features at the level of the...

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Replication: general features at the level of the whole chromosome • The circular E. Coli chromosome remains so during replication, forms a θ (theta) structure. • Replication starts at a specific place on the “circle” each time. • Evidence: in rapidly growing culture all genes are present at between 1 and 2 times the frequency of the “tre” gene. Those at 2 X “tre” are near the origin .
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Replication • Proceeds in both directions from the origin • Evidence: “label” DNA synthesis with moderately radioactive dTTP. Then switch to medium with highly radioactive dTTP. • Autoradiograph (lay a piece of X-ray or photographic film over layer of cells. Radiation exposes the film; causes spots on film) • Results: Darkly exposed regions occur on both sides of the lightly exposed one.
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Enzymes of replication • DNA ligase: joins the 3' OH on the end of one ssDNA segment to the 5' mono PO 4 on the end of another. • Uses the energy of ATP AMP + PP i (or, in E Coli, NAD + hydrolysis). • The two strands joined must each be base paired to a “template” in the region of joining: no gap, adjacent. (ligase “seals a nick” in double stranded DNA)
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Ligase
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Topoisomerase: changes DNA supercoiling Supercoiling is more or less twist (or bps per turn) than the DNA would have as a result of interactions within it and between it and the solution (i.e., differences from its “natural twist”). Type I relaxes supercoiling, does not need ATP (uses mechanical energy of supercoil). Type IA, present in all cells, relaxes negative supercoiling ; type IB relaxes positive or negative. A clue to the mechanism of type IA was the observation that it catenates (interlinks) and decatanates ssDNA circles.
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Topoisomerase Type IA • Type IA makes a single- strand break by attack of one of its tyrosyl side chain hydroxyls on a 5' PO 4 of the DNA. • The tyr side chain and the 5' PO 4 become covalently linked. • Then, the other ss DNA is “passed through” the break and the 5„ PO 4 is transferred back to the 3„ OH to which it was originally linked. • This unwinds one twist.
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Topoisomerase Type IA The mechanism begins with the tight binding of one of the strands in a groove in domain I. This places one of its PO 4 groups in proximity to the active site tyr, represented by the phe side chain F328 of domain III.
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Topoisomerase Type IA • Formation of the tyr-5‟ PO 4 linkage occurs along with a conformational change that moves domain III (which holds the 5‟ PO 4 ) away from domain I (which holds the ssDNA segment with the 3‟ OH ). • This creates a gap in the cut DNA strand (and in the enzyme) that the other DNA strand can pass through. • The 5‟ PO 4 is passed back to the 3‟ OH and the enzyme releases the product.
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Gyrase: a type II topoisomerase introduces negative supertwists using ATP --> ADP • also catenates and decatanates dsDNA circles • Negative supercoiling is required : • 1. to keep the E. Coli chromosome in compact form.
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This note was uploaded on 04/22/2011 for the course BIOCHEM 412 taught by Professor Thomas during the Spring '11 term at Tennessee Martin.

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412U3 - Replication: general features at the level of the...

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