Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1
Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1
复制标题
DOI：
10.1074/jbc.m109.010413
复制
发表时间：
2009-06-19
影响因子：
4.8
通讯作者：
Boehmer, Paul E.
中科院分区：
文献类型：
--
作者：
Bogani, Federica;Chua, Chian New;Boehmer, Paul E.
单纯疱疹病毒1型是一种大型双链DNA病毒，在许多基因组活动中具有自主性。因此，该病毒编码自身的DNA复制装置，并能够介导重组反应。我们最近报道，单纯疱疹病毒1型DNA聚合酶的催化亚基（UL30）具有无嘌呤/无嘧啶和5'-脱氧核糖磷酸裂解酶活性，这些活性是碱基切除修复所必需的。碱基切除修复是维持基因组稳定性所必需的，它是一种对抗异常碱基积累以及防止DNA碱基丢失的手段。在此，我们用纯化的单纯疱疹病毒1型蛋白和人类蛋白重建了一个系统，该系统可完成由尿嘧啶DNA糖基化酶启动的碱基切除修复的所有步骤。在这个系统中，核苷酸掺入依赖于单纯疱疹病毒1型尿嘧啶DNA糖基化酶（UL2）、人类AP内切核酸酶以及单纯疱疹病毒1型DNA聚合酶。碱基切除修复的完成可由T4 DNA连接酶以及人类DNA连接酶I或连接酶IIIα - XRCC1复合物介导。其中，连接酶IIIα - XRCC1效率最高。此外，连接酶IIIα - XRCC1赋予反应特异性，因为它允许在单纯疱疹病毒1型DNA聚合酶持续性因子（UL42）存在的情况下发生连接，并防止与异源DNA聚合酶发生碱基切除修复。在这个系统中，碱基切除修复的完成还依赖于正确核苷酸的掺入。这些发现表明，单纯疱疹病毒1型蛋白与病毒未编码的细胞因子结合能够进行碱基切除修复。这些结果对碱基切除修复在裂解复制和从潜伏期再激活过程中病毒基因组维持的作用具有启示意义。
Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase III alpha-XRCC1 complex. Of these, ligase III alpha-XRCC1 is the most efficient. Moreover, ligase III alpha-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.