Interplay between DNA Polymerase and Proliferating Cell Nuclear Antigen Switches Off Base Excision Repair of Uracil and Hypoxanthine during Replication in Archaea
Interplay between DNA Polymerase and Proliferating Cell Nuclear Antigen Switches Off Base Excision Repair of Uracil and Hypoxanthine during Replication in Archaea
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DOI：
10.1016/j.jmb.2008.08.018
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发表时间：
2008-11-21
影响因子：
5.6
通讯作者：
Connolly, Bernard A.
中科院分区：
文献类型：
--
作者：
Emptage, Kieran;O'Neill, Rory;Connolly, Bernard A.
古生菌B家族DNA聚合酶与尿嘧啶和次黄嘌呤(胞嘧啶和腺嘌呤的脱氨基产物)紧密结合，导致对DNA复制的严重抑制。用聚合酶超过DNA的单周转动力学对抑制机制进行了研究，结果表明脱氧NTPs能有效地结合到聚合酶-DNA复合体上，但很难结合到延伸链上。当尿嘧啶(K-d=16 Pm)或次黄嘌呤(Kd=65 Pm)存在时，加入过程性因子增殖细胞核抗原(PCNA)导致聚合酶对所有引物模板的亲和力增加，产生极其紧密的复合体。分析超速离心法证实了这些复合体的稳定性，并揭示了聚合酶/增殖细胞核抗原/DNA的化学计量比为1：1：1。然而，增殖细胞核抗原对聚合酶读取尿嘧啶和次黄嘌呤的能力没有影响，在有或没有加工因子的情况下观察到相同的动力学参数。用单周转动力学测定的特异性常数表明，尿嘧啶和次黄嘌呤分别使聚合酶减慢约5000倍和3000倍。尿嘧啶和次黄嘌呤分别由尿嘧啶-DNA糖基酶和核酸内切酶V启动，通过碱基切除修复从DNA中去除。这两种酶都被同时将增殖细胞核抗原和聚合酶结合到引子模板上而受到深刻的抑制，而仅有聚合酶的作用要小得多。因此，当增殖细胞核抗原-聚合酶复合体在DNA模板中遇到尿嘧啶/次黄嘌呤时，碱基切除修复被关闭，保护复合体免受修复途径的影响，而修复途径在复制过程中形成的单链DNA是危险的。(C)2008爱思唯尔有限公司。保留所有权利。
Archaeal family-B DNA polymerases bind tightly to uracil and hypoxanthine (the deamination products of cytosine and adenine), resulting in profound inhibition of DNA replication. Investigation of the mechanism of inhibition, using single-turnover kinetics with polymerase in excess of DNA, indicated that deoxy-NTPs were efficiently bound to the polymerase-DNA complex but very poorly incorporated into the extending chain. Addition of the processivity factor proliferating cell nuclear antigen (PCNA) resulted in increased affinity of the polymerase for all primer-templates, producing extremely tight complexes when uracil (K-d = 16 pM) or hypoxanthine (Kd = 65 pM) was present. Analytical ultracentrifugation confirmed the stability of these complexes and revealed a polymerase/PCNA/DNA stoichiometry of 1:1:1. However, PCNA had no influence on the ability of the polymerase to read through uracil and hypoxanthine, the same kinetic parameters being observed with or without the processivity factor. The specificity constants determined using single-turnover kinetics showed that uracil and hypoxanthine slowed the polymerase by factors of similar to 5000 and 3000, respectively. Uracil and hypoxanthine are removed from DNA by base excision repair, initiated by uracil-DNA glycosylase and endonuclease V, respectively. Both enzymes are profoundly inhibited by the simultaneous binding of both PCNA and polymerase to primer-templates, with polymerase alone being much less effective. Thus, when the PCNA-polymerase complex encounters uracil/hypoxanthine in DNA templates, base excision repair is switched off, protecting the complex from a repair pathway that is dangerous in the context of single-stranded DNA formed during replication. (C) 2008 Elsevier Ltd. All rights reserved.