Zhang Hong, Rhee Chanu, Bebenek Anna, Drake John W, Wang Jimin, Konigsberg William
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
Biochemistry. 2006 Feb 21;45(7):2211-20. doi: 10.1021/bi052099y.
Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.
新生碱基对结合口袋中存在单一位点替换的几种RB69 DNA聚合酶（RB69 pol）变体，与野生型酶相比，对非互补dNMP掺入的辨别能力较低。为了量化碱基选择性的损失，我们使用快速化学淬灭分析，测定了这些RB69 pol变体之一L561A的核酸外切酶缺陷形式掺入正确和所有错误dNMP组合的瞬态动力学参数。L561A变体对正确dNMP掺入的k(pol)和K(D)值没有显著改变，但相对于野生型酶，它显示出错配碱基掺入效率（k(pol)/K(D)）增加。L561A变体错配碱基的掺入效率范围从与模板C配对的dCMP的1.5×10^(-5) μM^(-1) s^(-1)到与模板C配对的dAMP的2×10^(-3) μM^(-1) s^(-1)。这些k(pol)/K(D)值比野生型酶观察到的值大3至60倍。通过用携带突变rII位点的T4噬菌体感染携带编码RB69 pol L561A变体质粒的大肠杆菌，测定L561A替换对体内突变频率的影响，并对rII(+)回复率进行评分。具有核酸外切酶活性的RB69 pol L561A表现出弱诱变表型。相反，用突变型或野生型T4噬菌体感染表达核酸外切酶缺陷型RB69 pol L561A的大肠杆菌后，没有产生子代噬菌体。这种显性致死表型归因于pol L561A和exo(-)诱变活性相结合预期的高突变率导致的错误灾难。
