TYPE-III RESTRICTION ENDONUCLEASES TRANSLOCATE DNA IN A REACTION DRIVEN BY RECOGNITION SITE-SPECIFIC ATP HYDROLYSIS

被引:110
作者
MEISEL, A
MACKELDANZ, P
BICKLE, TA
KRUGER, DH
SCHROEDER, C
机构
[1] UNIV BASEL, BIOZENTRUM, DEPT MICROBIOL, CH-4056 BASEL, SWITZERLAND
[2] HUMBOLDT UNIV BERLIN, MED SCH CHARITE, INST VIROL, D-10098 BERLIN, GERMANY
关键词
DNA-DEPENDENT ATPASE; DNA STRAND BIAS; DNA TRANSLOCATION; ECOP15I RECOGNITION SITE-SPECIFIC ATPASE; TYPE III DNA MODIFICATION AND RESTRICTION;
D O I
10.1002/j.1460-2075.1995.tb07296.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated, Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction, We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites, We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation, The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction, EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites, DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.
引用
收藏
页码:2958 / 2966
页数:9
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