Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains
Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains
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DOI：
10.1128/jb.00268-15
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发表时间：
2015-08-01
影响因子：
3.2
通讯作者：
Shuman, Stewart
中科院分区：
文献类型：
--
作者：
Jacewicz, Agata;Shuman, Stewart
耻垢分枝杆菌编码几种善于结合核糖核苷酸的DNA修复聚合酶，这就提出了DNA中的核糖核苷酸是如何被感知和去除的问题。耻毛分枝杆菌编码四种RNase H酶，是监视作用的有力候选。在这里，我们研究了耻垢分枝杆菌RnhC（一种双功能RNase H和酸性磷酸酶）的生化活性和核酸底物特异性。我们报道(i) RnhC核酸酶对RNA: DNA杂交双链具有严格特异性；（ii） RnhC不能选择性识别和切割双链核酸中的DNA-RNA或RNA-DNA连接；（iii） RnhC不能切割双链DNA中嵌入的单核糖核苷酸或二核糖核苷酸；（iv） RnhC可以切割嵌入双链DNA的4个或更多的核糖核苷酸束，在被切割的3'-OH端留下两个或更多的残余核糖核苷酸，在5-PO4端留下至少一个或两个核糖核苷酸；(v) RNase H活性固有于RnhC的140个氨基酸（aa） n端区域；(6) RnhC c端211-aa结构域是一种独立的酸性磷酸酶。RnhC的切割特异性与大肠杆菌RNase H2明显不同，后者选择性地在RNA-DNA连接处切割。因此，我们将RnhC归类为I型RNase h。RnhC的性质与冈崎片段RNA引物去除或嵌入DNA的寡核糖核苷酸束的监测作用一致，但与单个错误结合的核糖核苷酸的切除修复作用不一致。酶H有助于清除基因组中的核糖核苷酸，这些核糖核苷酸要么作为核糖（如RNA引物）存在，要么作为嵌入双链DNA中的单个核糖核苷酸存在。耻垢分枝杆菌编码四种RNase H蛋白，包括RnhC，这是本研究的特征。RnhC的核酸底物和切割位点特异性与启动核糖去除的作用一致，但在单核糖核苷酸监测中不一致。RnhC有一个c端酸性磷酸酶结构域，在功能上独立于其n端RNase H催化结构域。RnhC同源物在放线菌中普遍存在。
Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA: DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3'-OH end and at least one or two ribonucleotides on the 5-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides.IMPORTANCERNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. RnhC has a C-terminal acid phosphatase domain that is functionally autonomous of its N-terminal RNase H catalytic domain. RnhC homologs are prevalent in Actinobacteria.