Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII
Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII
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DOI：
10.1128/jb.00268-10
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发表时间：
2010-07-01
影响因子：
3.2
通讯作者：
Henneke, Ghislaine
中科院分区：
文献类型：
--
作者：
Le Laz, Sebastien;Le Goaziou, Audrey;Henneke, Ghislaine
在所有活着的生物体中，在连接新生DNA片段之前，忠实的DNA复制涉及从基因组DNA中去除RNA残基。由于古生菌2型核糖核酸酶H的生理作用还不完全清楚，因此需要进一步阐明检测核糖核酸酶H活性的底物结构要求。对深海热球菌基因组中检测到的单一核糖核酸酶H的生化特征表明，该2型核糖核酸酶H是一种依赖于镁和碱性pH的酶。PabRNase HII具有核糖核酸酶活性，是RNA-DNA/DNA双链上的一种特异性内切酶。这种特殊的裂解，在RNA-DNA连接的上游1个核苷酸，发生在一种底物上，在该底物中，RNA启动子必须完全退火到cdna模板。另一方面，5‘RNA瓣Okazaki片段中的PabRNase HII内切酶活性受损。此外，在靠近RNA-DNA连接的RNA部分引入错配降低了PabRNase HII切割的特异性和效率。此外，PabRNase HII可以切割嵌入在双链DNA中的单个核苷酸。我们的数据显示PabRNase HII是一种双功能酶，可能是完成DNA复制和DNA修复所必需的。
Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of Pyrococcus abyssi showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme. PabRNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA template. On the other hand, a 5' RNA flap Okazaki fragment intermediate impaired PabRNase HII endonuclease activity. Furthermore, introduction of mismatches into the RNA portion near the RNA-DNA junction decreased both the specificity and the efficiency of cleavage by PabRNase HII. Additionally, PabRNase HII could cleave a single ribonucleotide embedded in a double-stranded DNA. Our data revealed PabRNase HII as a dual-function enzyme likely required for the completion of DNA replication and DNA repair.