Analysis of nucleotide insertion and extension at 8-oxo-7,8-dihydroguanine by replicative T7 polymerase exo(-) and human immunodeficiency virus-1 reverse transcriptase using steady-state and pre-steady-state kinetics-猫眼课题宝

DOI：

10.1021/bi9627267

发表时间：

1997-05-27

期刊：

BIOCHEMISTRY

影响因子：

2.9

通讯作者：

中科院分区：

生物学3区

文献类型：

--

作者：

Furge, LL;Guengerich, FP

使用复制酶噬菌体聚合酶T7外切（-）（T7（-））和HIV-1逆转录酶（RT）检查了dCTP和dATP相对位点特异性8-氧代-7，8-二氢鸟嘌呤（8-oxoGua）（与dCTP插入相对G相反）掺入的前稳态动力学以及超出病变的延伸。将这些结果与大肠杆菌修复聚合酶I（KF-）和II（pol II-）exo（-）的先前发现进行比较[Lowe，L. G.，& Guengerich，F.（1996）Biochemistry 35，9840-9849]。HIV-1 RT显示出对dATP相对于8-oxoGua插入的非常高的偏好，其次是pol II-、T7（-）和KF-。稳态测定显示，对于相对于G或8-oxoGua插入dCTP和相对于8-oxoGua插入dATP，k（cat）始终低于稳态前聚合速率（k（p））。通过KF-、pol II-和T7（-）相对于8-oxoGua或G添加dCTP的预稳态动力学曲线都是双相的，具有快速的初始单转换爆发，随后是较慢的多转换速率，而通过这些聚合酶相对于8-oxoGua添加dATP没有显示爆发动力学。对于HIV-1 RT，相对于8-oxoGua添加dATP显示出爆发动力学，而添加dCTP则没有。通过硫代磷酸酯类似物取代正常dNTP的化学步骤的分析表明，在通过KF-、pol II-和T7（-）相对于8-oxoGua添加dCTP和dATP期间，化学是限速的; HIV-1 RT在相对于8-oxoGua添加dATP期间未显示化学限速步骤。用各种dCTP浓度进行的动力学测定表明，与G相比，对于所有四种酶，dCTP具有更高的K-d和更低的k（p），用于相对于8-氧代Gua掺入。在竞争测定中估计的KF-、pol II-和T7（-）掺入dATP相对于8-oxoGua的K-d，app（dATP）值比K-d（dCTP）大3-10倍。同样，发现dCTP相对于8-oxoGua的HIV-1 RT掺入的K-d，app（dCTP）比K-d（dATP）大10倍。（KF-和pol II-）有效地延长了8-oxoGua. A对; 8-oxoGua. C的延伸严重受损，而复制酶（T7（-）和HIV-1 RT）延伸两对，8-oxoGua.A对的延伸速率更快。在这些发现的基础上，在复制8-oxoGua的过程中，所有四种酶的保真度取决于表观Kd的贡献，碱基对延伸的容易程度，以及化学前的构象变化速率或键形成速率。

Pre-steady-state kinetics of incorporation of dCTP and dATP opposite site-specific 8-oxo-7,8-dihydroguanine (8-oxoGua), in contrast to dCTP insertion opposite G, were examined as well as extension beyond the lesion using the replicative enzymes bacteriophage polymerase T7 exo(-)(T7(-)) and HIV-1 reverse transcriptase (RT). These results were compared to previous findings for Escherichia coli repair polymerases I (KF-) and II (pol II-) exo(-) [Lowe, L. G., & Guengerich, F. P. (1996) Biochemistry 35, 9840-9849]. HIV-1 RT showed a very high preference for insertion of dATP opposite 8-oxoGua, followed by pol II-, T7(-), and KF-. Steady-state assays showed k(cat) consistently lower than pre-steady-state polymerization rates (k(p)) for insertion of dCTP opposite G or 8-oxoGua and insertion of dATP opposite 8-oxoGua. Pre-steady-state kinetic curves for the addition of dCTP opposite 8-oxoGua or G by KF-, pol II-, and T7(-) were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of dATP opposite 8-oxoGua by these polymerases did not display burst kinetics. With HIV-1 RT, addition of dATP opposite 8-oxoGua displayed burst kinetics while addition of dCTP did not. Analyses of the chemical step by substitution of phosphorothioate analogs for normal dNTPs suggest that the chemistry is rate-limiting during addition of dCTP and dATP opposite 8-oxoGua by KF-, pol II-, and T7(-); HIV-1 RT did not show a chemical rate-limiting step during addition of dATP opposite 8-oxoGua. Kinetic assays performed with various dCTP concentrations indicate that dCTP has a higher K-d and lower k(p) for incorporation opposite 8-oxoGua compared to G with all four enzymes. The K-d,app(dATP) values for KF-, pol II-, and T7(-) incorporation of dATP opposite 8-oxoGua, estimated in competition assays, were found to be 3-10-fold greater than the K-d(dCTP). Likewise, the K-d,app(dCTP) for HIV-1 RT incorporation of dCTP opposite 8-oxoGua was found to be 10-fold greater than the K-d(dATP) The repair enzymes (KF- and pol II-) efficiently extended the 8-oxoGua.A pair; extension of 8-oxoGua.C was severely impaired, whereas the replicative enzymes (T7(-) and HIV-1 RT) extended both pairs, with faster rates for the extension of the 8-oxoGua.A pair. On the basis of these findings, the fidelity of all four enzymes during replication of 8-oxoGua depends on contributions from the apparent K-d, the ease of base pair extension, and either the rate of conformational change before chemistry or the rate of bond formation.