Jayaram Sumithra, Ketner Gary, Adachi Noritaka, Hanakahi Les A
Department of Biochemistry and Molecular Biology, Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Nucleic Acids Res. 2008 Oct;36(18):5773-86. doi: 10.1093/nar/gkn552. Epub 2008 Sep 9.
The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.
通过非同源末端连接（NHEJ）修复DNA双链断裂对于维持基因组完整性和细胞活力至关重要。NHEJ分子机制的核心是DNA连接酶IV/XRCC4/XLF复合物，其可重新连接DNA。在腺病毒（Ad5）感染期间，连接酶IV在一个需要病毒E1B 55k和E4 34k蛋白表达的过程中被靶向降解，而XRCC4和XLF蛋白水平保持不变。我们发现，在Ad5感染的细胞中，连接酶IV的缺失伴随着XRCC4与DNA结合的丧失。E1B 55k和E4 34k的表达足以导致连接酶IV的丧失以及XRCC4与DNA结合的丧失。使用连接酶IV突变的人类细胞系，我们确定连接酶IV的缺失而非病毒蛋白的表达与XRCC4对DNA结合的抑制同时发生。在连接酶IV突变的人类细胞系中，XLF与DNA的结合也受到抑制。在连接酶IV缺陷的细胞中表达野生型和腺苷化突变型连接酶IV均可恢复XRCC4与DNA的结合。这些数据表明，XRCC4和XLF的内在DNA结合活性可能受到调控，并且在缺乏连接酶IV的人类细胞中被下调。
