Kerr S G, Anderson K S
Department of Pharmacology, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Biochemistry. 1997 Nov 18;36(46):14056-63. doi: 10.1021/bi971385+.
The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.
利用单周转条件下的前稳态动力学研究了HIV-1逆转录酶的RNA依赖性DNA复制保真度。与之前对HIV-1逆转录酶低复制保真度的估计相反，本研究发现，与相应的DNA/DNA模板引物相比，当使用RNA/DNA模板引物时，该酶具有更高的区分能力。这种选择性的基础是掺入错误脱氧核苷酸的聚合动力学极其缓慢。与模板尿苷相对的dGTP、dCTP和dTTP错误掺入的最大速率（kpol）分别为0.2、0.03和0.003 s-1。与模板尿苷相对的错误核苷酸的平衡解离常数（Kd），dGTP、dCTP和dTTP分别为1.0、1.1和0.7 mM。这些动力学值提供了在该位置针对dGTP错误掺入的保真度估计为26000，针对dCTP为176000，针对dTTP为1×10^6。当检查与模板腺嘌呤相对的错误核苷酸错误掺入时，也得到了类似的观察结果。因此，在RNA/DNA和DNA/DNA模板引物底物的直接比较中，HIV-1 RT的保真度提高了约10-60倍。这项研究加深了我们目前对HIV-1逆转录酶使用RNA和DNA底物时催化活性异同的理解。此外，这些研究进一步支持了HIV-1逆转录酶核苷酸掺入模型，该模型涉及一种两步结合机制，由正确掺入的限速构象变化控制。
