Analysis of the subunit assembly of the type IC restriction-modification enzyme EcoR124I
Analysis of the subunit assembly of the type IC restriction-modification enzyme EcoR124I
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DOI：
10.1093/nar/26.19.4439
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发表时间：
1998-10-01
影响因子：
14.9
通讯作者：
Firman, K
中科院分区：
文献类型：
--
作者：
Janscak, P;Dryden, DTF;Firman, K
I型限制修饰酶（R-M）由三个不同的亚基组成，其中HsdS决定DNA特异性，HsdM负责DNA甲基化，HsdR是限制所需的。HsdM和HsdS亚基也可以形成一个独立的DNA甲基转移酶，其亚基化学计量为M2 S1。我们发现，纯化的EcoR 1241 R-M酶是两种物质的混合物，这是通过凝胶阻滞试验中存在两种不同迁移的特异性DNA-蛋白质复合物来检测的。从复合物中分离的蛋白质亚基的分析表明，较大的种类具有R2 M2 S1的化学计量，较小的种类具有R1 M2 S1的化学计量。亚基组装的体外分析显示，虽然第一HsdR亚基与M2 S1复合物的结合非常紧密，第二个HsdR亚基与R1 M2 S1复合物的结合较弱，其表观Kd值约为2.4 × 10 ~（-7）M。功能测定表明，只有R2 M2 S1复合物能够切割DNA，然而，R1 M2 S1复合物保留ATP酶活性。这种情况的相关性进行了讨论，在体内的限制活性的调节后，接合转移的质粒出生的R-M系统到一个未修饰的宿主细胞。
Type I restriction-modification (R-M) enzymes are composed of three different subunits, of which HsdS determines DNA specificity, HsdM is responsible for DNA methylation and HsdR is required for restriction. The HsdM and HsdS subunits can also form an independent DNA methyltransferase with a subunit stoichiometry of M2S1, We found that the purified EcoR1241 R-M enzyme was a mixture of two species as detected by the presence of two differently migrating specific DNA-protein complexes in a gel retardation assay. An analysis of protein subunits isolated from the complexes indicated that the larger species had a stoichiometry of R2M2S1 and the smaller species had a stoichiometry of R1M2S1 In vitro analysis of subunit assembly revealed that while binding of the first HsdR subunit to the M2S1 complex was very tight, the second HsdR subunit was bound weakly and it dissociated from the R1M2S1 complex with an apparent K-d of similar to 2.4 x 10(-7) M. Functional assays have shown that only the R2M2S1 complex is capable of DNA cleavage, however, the R1M2S1 complex retains ATPase activity. The relevance of this situation is discussed in terms of the regulation of restriction activity in vivo upon conjugative transfer of a plasmid-born R-M system into an unmodified host cell.