Bibillo Arkadiusz, Lener Daniela, Tewari Alok, Le Grice Stuart F J
Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI, Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
J Biol Chem. 2005 Aug 26;280(34):30282-90. doi: 10.1074/jbc.M502457200. Epub 2005 Jun 7.
Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.
氨基酸序列比对被用于鉴定酿酒酵母中含长末端重复序列的逆转录转座子Ty3的逆转录酶（RT）的推定拇指亚结构域。在Ty3酶的第290至298位氨基酸之间鉴定出了与人类免疫缺陷病毒1型（HIV-1）RT的αH螺旋相对应的结构，该螺旋介导了与DNA聚合酶催化中心后约3 - 6个碱基对处的双链核酸的重要相互作用。研究了用Ty3 RT的Gln290、Phe292、Gly294、Asn297和Tyr298（分别对应HIV-1 RT的Gln258、Leu260、Gly262、Asn265和Trp266）替代对DNA聚合酶和核糖核酸酶H活性的影响。在未修饰的底物以及模板或引物中含有锁定核酸类似物靶向插入和无碱基损伤的双链体上评估了依赖DNA的DNA合成。基于这种联合策略，我们的数据表明Ty3 RT的Tyr298与引物核苷酸-3相互作用，Gly294与引物核苷酸-4相互作用，Asn297与模板核苷酸-6相互作用。尽管该位置具有高度保守性，但用丙氨酸替代Gln290具有良好的耐受性。Ty3拇指亚结构域的突变也影响了核糖核酸酶H活性，这表明与HIV-1 RT相比，其N端和C端催化中心之间的空间关系更紧密。
