Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation
Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation
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DOI：
10.1016/j.bbrc.2014.10.044
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发表时间：
2014-11-14
影响因子：
3.1
通讯作者：
Yasukawa, Kiyoshi
中科院分区：
文献类型：
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作者：
Konishi, Atsushi;Hisayoshi, Tetsuro;Yasukawa, Kiyoshi
我们先前已经使用定点诱变来引入碱性残基（即，Arg;在Moloney鼠白血病病毒逆转录酶（MMLV RT）的核酸结合裂缝中添加赖氨酸（Lys）以增加其模板-引物（T/P）结合亲和力。三个稳定突变（即，E286 R、E302 K和L435 R）（Yasukawa等人，2010年）。现在，我们研究了这些突变增加RT热稳定性的机制。在大肠杆菌中产生了三种单突变体（E286 R、E302 K和L435 R）、RNase H缺陷型MMLV RT（携带RNase H失活突变D524 A）、四重突变体（E286 R/E302 K/L435 R/D524 A，指定为MM 4）和野生型酶（WT）。所有RT对杂聚DNA/DNA（2.9-6.5 nM）和RNA/DNA复合物（1.2-2.9 nM）显示出相似的解离常数（K-d）。与WT不同，突变酶（E286 R、E302 K、L435 R、D524 A和MM 4）缺乏RNA酶H活性，并且不能降解RNA/DNA复合物中的RNA。这些结果表明，突变，E286 R，E302 K，和L435 R增加MMLV RT的热稳定性不是通过增加其对T/P的亲和力，而是通过消除其RNase H活性。(C)2014 Elsevier Inc. All rights reserved.
We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (K-d) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity. (C) 2014 Elsevier Inc. All rights reserved.