Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication
Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication
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DOI：
10.1006/jmbi.2001.4494
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发表时间：
2001-03-23
影响因子：
5.6
通讯作者：
Tainer, JA
中科院分区：
文献类型：
--
作者：
Chapados, BR;Chai, Q;Tainer, JA
DNA复制和细胞存活需要在滞后链DNA合成期间有效去除RNA引物。在真核生物中，RNA引物去除由2型RNA酶H启动，其特异性切割RNA-DNA/DNA杂交双链体的RNA部分。这种保守的复制酶的2型RNase H家族与充分表征的原核1型RNase H酶几乎没有序列相似性，但两者都具有相似的酶性质。晶体结构和基于结构的突变分析的RNA酶HII从古生球菌fulgidus，有和没有结合的金属离子，确定2型RNA酶H酶的活性位点，提供催化所需的一般核酸酶活性。2型RNA酶H的双结构域结构产生带正电荷的结合沟，并将独特的C-末端螺旋-环-螺旋帽结构域连接到活性位点催化结构域。这种结构安排显然耦合定向A型双链体结合，通过氢键Arg-Lys磷酸统治者基序，底物歧视，通过酪氨酸指状基序，从而提供底物特异性催化活性。结合动力学和突变分析的结构牵连底物结合残基验证这种结合模式。这些结构和突变结果共同表明，2型RNA酶H酶特异性识别和切割杂交双链体内RNA-DNA连接处的RNA的分子机制，这使广泛的底物结合亲和力与生化测定中观察到的催化特异性相协调。结合最近的独立结构分析，这些结果进一步确定了2型RNA酶H家族酶的活性和功能的可测试的分子假设，包括结构互补性，底物介导的构象变化和与随后的FEN-1活性的协调。(C)北京：科学出版社.
DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. Ln combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity. (C) 2001 Academic Press.