Sasakawa Noriko, Fukui Tomoyuki, Waga Shou
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043.
J Biochem. 2006 Jul;140(1):95-103. doi: 10.1093/jb/mvj130. Epub 2006 Jun 23.
Werner syndrome is a genetic disorder characterized by premature aging and cancer-prone symptoms, and is caused by mutation of the WRN gene. WRN is a member of the RecQ helicase family and is thought to function in processes implicated in DNA replication and repair to maintain genome stability; however, its precise function is still unclear. We found that replication fork arrest markedly enhances chromatin binding of focus-forming activity 1 (FFA-1), a Xenopus WRN homolog, in Xenopus egg extracts. In addition to FFA-1, DNA polymerase delta (Poldelta) and replication protein A, but not DNA polymerase epsilon and proliferating cell nuclear antigen, accumulated increasingly on replication-arrested chromatin. Elevated accumulation of these proteins was dependent on formation of pre-replicative complexes (pre-RCs). Double-strand break (DSB) formation also enhanced chromatin binding of FFA-1, but not Poldelta, independently of pre-RC formation. In contrast to FFA-1, chromatin binding of Xenopus Bloom syndrome helicase (xBLM) only slightly increased after replication arrest or DSB formation. Thus, WRN-specific, distinct processes can be reproduced in the in vitro system in egg extracts, and this system is useful for biochemical analysis of WRN functions during DNA metabolism.
沃纳综合征是一种遗传性疾病，其特征为早衰和易患癌症的症状，由WRN基因突变引起。WRN是RecQ解旋酶家族的成员，被认为在与DNA复制和修复相关的过程中发挥作用以维持基因组稳定性；然而，其确切功能仍不清楚。我们发现，在非洲爪蟾卵提取物中，复制叉停滞会显著增强非洲爪蟾WRN同源物聚焦形成活性1（FFA-1）与染色质的结合。除了FFA-1，DNA聚合酶δ（Poldelta）和复制蛋白A，但不是DNA聚合酶ε和增殖细胞核抗原，在复制停滞的染色质上逐渐积累。这些蛋白质的积累增加依赖于复制前复合物（pre-RCs）的形成。双链断裂（DSB）的形成也增强了FFA-1与染色质的结合，但不影响Poldelta，且与pre-RC形成无关。与FFA-1不同，非洲爪蟾布鲁姆综合征解旋酶（xBLM）与染色质的结合在复制停滞或DSB形成后仅略有增加。因此，WRN特异性的、独特的过程可以在卵提取物的体外系统中重现，该系统对于DNA代谢过程中WRN功能的生化分析很有用。
