Restriction glycosylases: involvement of endonuclease activities in the restriction process.
Restriction glycosylases: involvement of endonuclease activities in the restriction process.
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DOI：
10.1093/nar/gkw1250
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发表时间：
2017-02-17
影响因子：
14.9
通讯作者：
Kobayashi I
中科院分区：
文献类型：
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作者：
Zhang Y;Matsuzaka T;Yano H;Furuta Y;Nakano T;Ishikawa K;Fukuyo M;Takahashi N;Suzuki Y;Sugano S;Ide H;Kobayashi I
所有检测的限制性内切酶都是产生3个β-OH和5个β-P末端的磷酸二酯酶，但有一种限制性内切酶（限制性糖基化酶）从其识别序列中切除未甲基化的碱基。其限制活性是否涉及核酸内切酶切仍不清楚。关于这种酶的一份报告，来自超嗜热菌的R.PabI，将断裂归因于高温，而另一份报告显示其弱AP裂解酶活性产生非典型末端。在这里，我们解决了这个问题在嗜温细胞。我们从大肠弯曲杆菌（R.CcoLI）和幽门螺杆菌（R.HpyAXII）中纯化了R.PabI同源物，并在37°C下体外证明了它们的DNA切割、DNA糖基化酶和AP裂解酶活性。R.CcoLI中AP裂解酶活性与糖基化酶活性的耦合程度高于R. PabI。R.CcoLI/R.PabI表达在37°C下引起大肠杆菌内进入的噬菌体/质粒DNA和内源性染色体DNA的限制。R. PabI介导的限制性酶切在体内或体外由AP核酸内切酶作用促进。这些结果揭示了核酸内切DNA切割在限制性内切酶中的作用，并指出了核酸内切酶之间的多样性。切割的末端在体内难以修复，这可能表明它们的生物学意义。这些结果支持将限制修饰系统的概念推广到自我识别表观遗传系统的概念，其结合了任何表观遗传标记和任何DNA损伤。
All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction–modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.