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Dynamics of DNA loop capture by the SfiI restriction endonuclease on supercoiled and relaxed DNA.

Abstract：

:The SfiI endonuclease is a prototype for DNA looping. It binds two copies of its recognition sequence and, if Mg(2+) is present, cuts both concertedly. Looping was examined here on supercoiled and relaxed forms of a 5.5 kb plasmid with three SfiI sites: sites 1 and 2 were separated by 0.4 kb, and sites 2 and 3 by 2.0 kb. SfiI converted this plasmid directly to the products cut at all three sites, though DNA species cleaved at one or two sites were formed transiently during a burst phase. The burst revealed three sets of doubly cut products, corresponding to the three possible pairings of sites. The equilibrium distribution between the different loops was evaluated from the burst phases of reactions initiated by adding MgCl(2) to SfiI bound to the plasmid. The short loop was favored over the longer loops, particularly on supercoiled DNA. The relative rates for loop capture were assessed after adding SfiI to solutions containing the plasmid and MgCl(2). On both supercoiled and relaxed DNA, the rate of loop capture across 0.4 kb was only marginally faster than over 2.0 kb or 2.4 kb. The relative strengths and rates of looping were compared to computer simulations of conformational fluctuations in DNA. The simulations concurred broadly with the experimental data, though they predicted that increasing site separations should cause a shallower decline in the equilibrium constants than was observed but a slightly steeper decline in the rates for loop capture. Possible reasons for these discrepancies are discussed.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Embleton ML,Vologodskii AV,Halford SE

doi

10.1016/j.jmb.2004.03.046

keywords:

subject

Has Abstract

pub_date

2004-05-21 00:00:00

pages

53-66

issue

1

eissn

0022-2836

issn

1089-8638

pii

S0022283604003481

journal_volume

339

pub_type

杂志文章

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