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Functional characterization and modulation of the DNA cleavage efficiency of type III restriction endonuclease EcoP15I in its interaction with two sites in the DNA target.

Abstract：

:EcoP15I is a Type III restriction endonuclease requiring the interaction with two inversely oriented 5'-CAGCAG recognition sites for efficient DNA cleavage. Diverse models have been developed to explain how enzyme complexes bound to both sites move toward each other, DNA translocation, DNA looping and simple diffusion along the DNA. Conflicting data also exist about the impact of cofactor S-adenosyl-L-methionine (AdoMet), the AdoMet analogue sinefungin and the bases flanking the DNA recognition sequence on EcoP15I enzyme activity. To clarify the functional role of these questionable parameters on EcoP15I activity and to optimize the enzymatic reaction, we investigated the influence of cofactors, ionic conditions, bases flanking the recognition sequence and enzyme concentration. We found that AdoMet is not necessary for DNA cleavage. Moreover, the presence of AdoMet dramatically impaired DNA cleavage due to competing DNA methylation. Sinefungin neither had an appreciable effect on DNA cleavage by EcoP15I nor compensated for the second recognition site. Moreover, we discovered that adenine stretches on the 5' or 3' side of CAGCAG led to preferred cleavage of this site. The length of the adenine stretch was pivotal and had to be different on the two sides for most efficient cleavage. In the absence of AdoMet and with enzyme in molar excess over recognition sites, we observed minor cleavage at two communicating DNA sites simultaneously. These results could also be exploited in the high-throughput, quantitative transcriptome analysis method SuperSAGE to optimize the crucial EcoP15I digestion step.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Möncke-Buchner E,Rothenberg M,Reich S,Wagenführ K,Matsumura H,Terauchi R,Krüger DH,Reuter M

doi

10.1016/j.jmb.2009.02.047

subject

Has Abstract

pub_date

2009-04-17 00:00:00

pages

1309-19

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(09)00218-6

journal_volume

387

pub_type

杂志文章

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Functional characterization and modulation of the DNA cleavage efficiency of type III restriction endonuclease EcoP15I in its interaction with two sites in the DNA target.