Douglas Max E, Diffley John F X
From The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
From The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
J Biol Chem. 2016 Mar 11;291(11):5879-5888. doi: 10.1074/jbc.M115.707802. Epub 2015 Dec 30.
Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly ("G1-like") and high affinity recruitment when CMG assembly takes place ("S-phase-like"). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus.
Mcm10是真核生物DNA复制起始所必需的，并且以某种未知方式促进Cdc45-MCM-GINS（CMG）解旋酶的激活。目前尚不清楚Mcm10如何定位于复制起始位点，因为当前模型表明与微小染色体维持蛋白（MCM）的直接结合起作用，但这种相互作用的细节和功能重要性尚未确定。在这里，我们表明纯化的Mcm10可以结合与DNA结合的MCM双六聚体和可溶性单六聚体。Mcm10与MCM的结合需要Mcm10的C末端。此外，Mcm10在MCM上的结合位点包括Mcm2和Mcm6亚基，并且与装载因子Cdt1的结合位点重叠。目前尚不清楚Mcm10募集到复制起点是否取决于CMG解旋酶的组装。我们表明Mcm10的募集通过两种模式发生：在没有CMG组装的情况下的低亲和力募集（“G1期样”）和在CMG组装发生时的高亲和力募集（“S期样”）。不能直接与MCM结合的Mcm10在两种募集模式中均有缺陷，并且不能支持DNA复制。这些发现表明Mcm10通过Mcm10的C末端直接结合MCM而定位于复制起始位点。
