Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks
Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks
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二聚 CMG/预起始复合物的表征及其向 DNA 复制叉的转变
DOI：
10.1007/s00018-019-03333-9
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发表时间：
2020
影响因子：
8
通讯作者：
Lou Huiqiang
中科院分区：
文献类型：
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作者：
Liu Lu;Zhang Yue;Zhang Jingjing;Wang Jian-Hua;Cao Qinhong;Li Zhen;Campbell Judith L.;Dong Meng-Qiu;Lou Huiqiang
前起始复合体(Pre-IC)作为真核生物DNA复制叉成熟前的中间体已经被提出了二十年。然而，它的存在和生化性质仍然是个谜。在这里，通过结合几种浓缩策略，我们能够分离出不同于传统的单一CMG(S-CMG)和体外重组的二聚体CMG的内源性二聚体CMG复合体(称为d-CMG)。D-CMG在进入S时组装，并很快成熟为S-CMG/复制体，导致在体内只有5%的含CMG的复合体可被检测到d-CMG。质谱图显示RPA和DNAolα/Primase与S-CMG协同纯化，但不与d-CMG协同纯化。始终如一的是，前者能够催化DNA解离和从头合成，而后者两者都不催化。D-CMG中的两个CMG在电子显微镜下显示出灵活的取向构象。当DNAPolα-Primase失活时，d-CmG%上升到29%，表明前IC/Fork转换不完整。这些发现揭示了d-CMG/Pre-IC的生化特性，并提供了体内证据，支持Pre-IC/Fork转变是复制启动的真正步骤。
The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona fide step in replication initiation.