The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB
The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB
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DOI：
10.1515/bchm.1997.378.9.975
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发表时间：
1997-09-01
影响因子：
3.7
通讯作者：
Pingoud, A
中科院分区：
文献类型：
--
作者：
Gast, FU;Brinkmann, T;Pingoud, A
McrBC 是大肠杆菌 K12 的 GTP 依赖性限制性内切酶，选择性针对含有修饰胞嘧啶残基的 DNA，McrB（其组分之一）负责结合，并与 McrC 一起裂解含有两个 5'-(PuC)-C-m 位点（间隔 40-80 个碱基对）的 DNA。使用野生型和突变型 McrB 进行的凝胶阻滞测定表明 (i) DNA 中的单个 5'-(PuC)-C-m 位点足以引发 McrB 的结合，然而，与此类底物的结合很弱并且强烈依赖于 (PuC)-C-m 位点的序列背景，(ii) 强 DNA 结合（K-ass 类似于 10(7) M-1）取决于至少 两个 (PuC)-C-m 位点，即使它们相距小于 40 bp，并且受序列上下文调节 (-A(m)CCGGT->-A(m)CT(C)/(G)AGT->-AGG(m)CCT->-AAG(m)CTT-)，(iii) McrB 的 DNA 结合伴随着不同的多重复合物的形成，其分布受 GTP 调节， (iv) McrC 本身不能结合 DNA，它会适度刺激 McrB 的 DNA 结合，并将 McrB-DNA 复合物转化为大聚集体，(v) McrB 的 C 末端一半（其包含鸟嘌呤核苷酸结合蛋白的三个共有序列特征）的缺失，导致蛋白质在 GTP 结合和/或水解中失活，并在 McrC 辅助 DNA 切割；然而，该蛋白质仍然完全具有 DNA 结合能力。 (vi) McrB 中的突变表明 GTP 结合和/或水解减少，可以影响 DNA 结合，表明这两种活性在全长蛋白质中耦合。
McrBC is a GTP-dependent restriction endonuclease of E, coli K12, selectively directed against DNA containing modified cytosine residues, McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-(PuC)-C-m sites separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-(PuC)-C-m sites in DNA can be sufficient to elicite binding by McrB, Binding to such substrates is, however, weak and strongly dependent on the sequence context of (PuC)-C-m sites, (ii) Strong DNA binding (K-ass similar to 10(7) M-1) is dependent on the presence of at least two (PuC)-C-m sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT->-A(m)CT(C)/(G)AGT->-AGG(m)CCT->-AAG(m)CTT-), (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP, (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates, (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding, (vi) Mutations in McrB which read to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.