Silverman Adam P, Garforth Scott J, Prasad Vinayaka R, Kool Eric T
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Biochemistry. 2008 Apr 22;47(16):4800-7. doi: 10.1021/bi702427y. Epub 2008 Mar 27.
The steric flexibility or rigidity of polymerase active sites may play an important role in their fidelity of nucleic acid synthesis. In this regard, reverse transcriptases offer an unusual opportunity to compare two enzymatic activities that proceed in the same active site. For HIV-1 reverse transcriptase, reverse transcription (RNA-templated synthesis) is known to proceed with lower fidelity than DNA-templated synthesis. Here, we describe the use of a set of variably sized nonpolar thymidine and uracil mimics as molecular rulers to probe the active site steric constraints of HIV-1 RT, and for the first time, we directly compare the functional flexibility of these two activities. Steady-state kinetics of incorporation for natural dNTPs opposite unnatural template bases as well as for unnatural dNTPs opposite natural template bases are reported for the DNA-templated DNA synthesis, and comparison is made with recent data for the RNA-templated activity. Kinetics for extension beyond a base pair containing the analogue template bases are also reported both for RNA and DNA templates. Our results show that the DNA-dependent polymerization by HIV-RT is highly sensitive to size, strongly biasing against both too-small and too-large base pairs, while, by contrast, the RNA-dependent polymerization is only biased against analogues that are too small, and is much more accepting of larger base pairs. In addition, base pair extension with HIV-RT is found to be relatively insensitive to varied base pair size, consistent with its high mutagenicity. Overall, the data show greater rigidity with a DNA template as compared with an RNA template, which correlates directly with the higher fidelity of the DNA-templated synthesis. Possible structural explanations for these differences are discussed. We also report kinetics data for two HIV-1 RT mutants reported to have altered fidelity (F61A and K65R) using DNA templates containing nonpolar base analogues, and find that one of these (F61A) is a high-fidelity enzyme that appears to be sensitive to a loss of hydrogen-bonding groups.
聚合酶活性位点的空间灵活性或刚性可能在其核酸合成保真度方面发挥重要作用。在这方面，逆转录酶提供了一个独特的机会来比较在同一活性位点进行的两种酶促活性。对于HIV-1逆转录酶，已知逆转录（以RNA为模板的合成）的保真度低于以DNA为模板的合成。在此，我们描述了使用一组大小可变的非极性胸腺嘧啶和尿嘧啶类似物作为分子尺来探测HIV-1 RT的活性位点空间限制，并且首次直接比较这两种活性的功能灵活性。报道了以非天然模板碱基相对的天然dNTP掺入以及以天然模板碱基相对的非天然dNTP掺入的稳态动力学，用于以DNA为模板的DNA合成，并与最近关于以RNA为模板活性的数据进行了比较。还报道了对于RNA和DNA模板，延伸超过包含类似物模板碱基的碱基对的动力学。我们的结果表明，HIV-RT的依赖DNA的聚合对大小高度敏感，强烈偏向于既不过小也不过大的碱基对，而相比之下，依赖RNA的聚合仅偏向于过小的类似物，并且对更大的碱基对更能接受。此外，发现HIV-RT的碱基对延伸对不同的碱基对大小相对不敏感，这与其高致突变性一致。总体而言，数据表明与RNA模板相比，DNA模板具有更大的刚性，这与以DNA为模板的合成的更高保真度直接相关。讨论了这些差异可能的结构解释。我们还报道了使用含有非极性碱基类似物的DNA模板对两个据报道具有改变的保真度的HIV-1 RT突变体（F61A和K65R）的动力学数据，并发现其中一个（F61A）是一种高保真酶，似乎对氢键基团的丧失敏感。
