Matsuno Kumiko, Kumano Maya, Kubota Yumiko, Hashimoto Yoshitami, Takisawa Haruhiko
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Mol Cell Biol. 2006 Jul;26(13):4843-52. doi: 10.1128/MCB.02267-05.
Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases alpha and epsilon onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase alpha. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.
将DNA聚合酶招募到复制起点是真核生物复制机器组装过程中的关键一步。先前对芽殖酵母的一项研究表明，Dpb11控制DNA聚合酶α和ε向起点的招募。Sld2是一种与Dpb11相互作用的必需复制蛋白，但尚未鉴定出后生动物同源物。我们分离出非洲爪蟾RecQ4作为候选的Sld2同源物。RecQ4是后生动物RecQ解旋酶家族的成员，其N端区域与Sld2具有序列相似性。在非洲爪蟾卵提取物中，RecQ4对DNA复制的起始至关重要，特别是对DNA聚合酶α的染色质结合。缺失解旋酶结构域的RecQ4的N端片段可以挽救RecQ4缺失提取物的复制活性，并且针对该片段的抗体抑制DNA复制和聚合酶的染色质结合。此外，RecQ4的N端片段与Dpb11的非洲爪蟾同源物Cut5发生物理相互作用，它们与Cut5结合的能力与其挽救缺失提取物复制活性的能力密切相关。我们的数据表明，RecQ4在脊椎动物中通过与Cut5相互作用在复制机器的组装中发挥重要作用。
