Archaeoglobus fulgidus RNase HII in DNA replication:: Enzymological functions and activity regulation via metal cofactors
Archaeoglobus fulgidus RNase HII in DNA replication:: Enzymological functions and activity regulation via metal cofactors
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DOI：
10.1006/bbrc.2001.5523
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发表时间：
2001-09-07
影响因子：
3.1
通讯作者：
Shen, BH
中科院分区：
文献类型：
--
作者：
Chai, Q;Qiu, J;Shen, BH
DNA复制过程中RNA引物的去除依赖于核糖核苷酸特异性和结构特异性RNase H和FEN-1核酸酶活性。长期以来，人们一直在寻找参与该反应的特异性RNase H。RNase H Ⅱ是闪烁古生菌基因组中唯一的开放阅读框架，而在真核细胞中存在多种RNase H。本文提供的数据表明，来自A.荧光素酶（aRNase HII）特异性识别RNA-DNA连接并产生适合于FEN-1核酸酶的产物，表明其在DNA复制中的作用。在各种二价金属离子存在下aRNase HII活性的生物化学表征揭示了对Mg 2+和Mn 2+具有偏好的宽金属耐受性。结合诱变，生化竞争，和金属依赖性活性测定进一步澄清底物结合或催化，分别确定的氨基酸残基的功能。这些实验还表明，Asp 129形成第二金属结合位点，从而有助于活性衰减。(C)北京：科学出版社.
RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA-DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg2+ and Mn2+. Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation. (C) 2001 Academic Press.