Gu J, Villanueva R A, Snyder C S, Roth M J, Georgiadis M M
Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
J Mol Biol. 2001 Jan 12;305(2):341-59. doi: 10.1006/jmbi.2000.4281.
Reverse transcriptase, an essential retroviral DNA polymerase, replicates the single-stranded RNA genome of the retrovirus, producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. Substitution of Ala for either Asp114 or Arg116, two highly conserved residues in the fingers domain of Moloney murine leukemia virus reverse transcriptase, results in enzymes (D114A or R116A) with significant defects in their abilities to processively synthesize DNA using RNA or DNA as a template. D114A and R116A enzymes also bind more weakly to template-primer in the presence of added deoxyribonucleotides, as seen by gel-shift analysis, but retain the ability to strand transfer and accumulate smaller RNase H cleavage products when compared to the wild-type enzyme. In addition, mutant proviruses, including D114A and R116A substitutions in Moloney murine leukemia virus reverse transcriptase, are not viable despite the presence of processed reverse transcriptase in the viral particles. A potential mechanistic role in processive synthesis for D114 and R116 is discussed in the context of our results, related studies on HIV-1 reverse transcriptase, and previous structural studies.
逆转录酶是一种必不可少的逆转录病毒DNA聚合酶,它复制逆转录病毒的单链RNA基因组,产生双链DNA拷贝,随后该拷贝整合到宿主基因组中。用丙氨酸取代莫洛尼鼠白血病病毒逆转录酶指状结构域中两个高度保守的残基天冬氨酸114或精氨酸116,会导致酶(D114A或R116A)在以RNA或DNA为模板进行连续DNA合成的能力上存在显著缺陷。凝胶迁移分析表明,在添加脱氧核糖核苷酸的情况下,D114A和R116A酶与模板引物的结合也更弱,但与野生型酶相比,它们保留了链转移能力并积累较小的核糖核酸酶H切割产物。此外,尽管病毒颗粒中存在经过加工的逆转录酶,但包括莫洛尼鼠白血病病毒逆转录酶中D114A和R116A取代在内的突变前病毒无法存活。我们结合研究结果、关于HIV-1逆转录酶的相关研究以及之前的结构研究,讨论了D114和R116在连续合成中的潜在机制作用。