Characterization of the uracil-DNA glycosylase activity of Epstein-Barr virus BKRF3 and its role in lytic viral DNA replication
Characterization of the uracil-DNA glycosylase activity of Epstein-Barr virus BKRF3 and its role in lytic viral DNA replication
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DOI：
10.1128/jvi.01518-06
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发表时间：
2007-02-01
影响因子：
5.4
通讯作者：
Chen, Mei-Ru
中科院分区：
文献类型：
--
作者：
Lu, Chih-Chung;Huang, Ho-Ting;Chen, Mei-Ru
尿嘧啶-N-糖基化酶（UNG）家族的尿嘧啶-DNA糖基化酶（UDG）是负责从DNA中去除不适当的尿嘧啶的主要DNA修复酶。最近的研究进一步表明，核人类UNG 2和大DNA病毒的UDG可能与它们的DNA聚合酶辅助因子协调以增强DNA复制。基于其氨基酸序列，EB病毒（EBV）的推定UDG BKRF 3属于UNG蛋白家族，并且先前在共转染复制测定中证明其增强oriLy依赖性DNA复制。然而，EBV BKRF 3的表达和酶活性尚未被表征。在这项研究中，His-BKRF 3在细菌中表达并纯化用于生化分析。与大肠杆菌和人UNG酶的情况类似，His-BKRF 3从单链DNA比从双链DNA更有效地切除尿嘧啶，并且被纯化的噬菌体PBS 1抑制剂Ugi抑制。此外，BKRF 3能够与E.在利福平和萘啶酸抗性突变体测定中，用BKRF 3特异性小鼠抗体检测BKRF 3产物在EBV阳性淋巴样细胞和上皮细胞中的表达动力学和亚细胞定位。BKRF 3的表达主要受即刻早期转录激活因子Rta的调控。EBV裂解DNA复制的效率受到BKRF 3小干扰RNA（siRNA）的轻微影响，而细胞UNG 2 siRNA或通过表达Ugi抑制细胞和病毒UNG活性抑制EBV裂解DNA复制。综合这些结果，我们证明了BKRF 3在体外和体内的UNG活性，并表明UNG可能参与DNA复制或修复，从而促进病毒DNA的有效生产。
Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.