Sangrithi Mahesh N, Bernal Juan A, Madine Mark, Philpott Anna, Lee Joon, Dunphy William G, Venkitaraman Ashok R
Cancer Research UK Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, United Kingdom.
Cell. 2005 Jun 17;121(6):887-98. doi: 10.1016/j.cell.2005.05.015.
How the replication machinery is loaded at origins of DNA replication is poorly understood. Here, we implicate in this process the Xenopus laevis homolog (xRTS) of the RECQL4 helicase mutated in Rothmund-Thomson syndrome. xRTS, which bears homology to the yeast replication factors Sld2/DRC1, is essential for DNA replication in egg extracts. xRTS can be replaced in extracts by its human homolog, while RECQL4 depletion from mammalian cells induces proliferation failure, suggesting an evolutionarily conserved function. xRTS accumulates on chromatin during replication initiation, after prereplication-complex (pre-RC) proteins, Cut5, Sld5, or Cdc45 but before replicative polymerases. xRTS depletion suppresses the loading of RPA, the ssDNA binding protein that marks unwound origins before polymerase recruitment. However, xRTS is unaffected by xRPA depletion. Thus, xRTS functions after pre-RC formation to promote loading of replication factors at origins, a previously unrecognized activity necessary for initiation. This role connects defective replication initiation to a chromosome-fragility disorder.
DNA复制起始位点处的复制机制是如何被装载的，目前还知之甚少。在此，我们发现非洲爪蟾（Xenopus laevis）中与在罗思蒙德-汤姆森综合征（Rothmund-Thomson syndrome）中发生突变的RECQL4解旋酶同源的蛋白（xRTS）参与了这一过程。xRTS与酵母复制因子Sld2/DRC1具有同源性，对于卵提取物中的DNA复制至关重要。在提取物中，xRTS可以被其人类同源物替代，而从哺乳动物细胞中去除RECQL4会导致增殖失败，这表明其具有进化上保守的功能。在复制起始过程中，xRTS在复制前复合体（pre-RC）蛋白Cut5、Sld5或Cdc45之后，但在复制性聚合酶之前，在染色质上积累。xRTS的缺失会抑制RPA的装载，RPA是一种单链DNA结合蛋白，在聚合酶募集之前标记解开的起始位点。然而，xRTS不受xRPA缺失的影响。因此，xRTS在pre-RC形成后发挥作用，以促进复制因子在起始位点的装载，这是起始过程中一种以前未被认识到的必要活性。这一作用将有缺陷的复制起始与一种染色体脆性疾病联系起来。
