Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme.

Abstract:

:Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25-27 bp downstream of one of the two sites. The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation. The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme. In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site. Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site. Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates. Such cleavage can be abolished by the binding of Lac repressor downstream (3' side) but not upstream (5' side) of the recognition site. Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity. In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5' to 3' direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends. This study demonstrates a new facet in the mode of action of these restriction enzymes.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Raghavendra NK,Rao DN

doi

10.1093/nar/gkh899

keywords:

subject

Has Abstract

pub_date

2004-10-22 00:00:00

pages

5703-11

issue

19

eissn

0305-1048

issn

1362-4962

pii

32/19/5703

journal_volume

32

pub_type

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