Perez-Arnaiz Patricia, Bruck Irina, Colbert Max K, Kaplan Daniel L
Florida State University College of Medicine, Department of Biomedical Sciences, Tallahassee, FL 32306, USA.
Nucleic Acids Res. 2017 Jul 7;45(12):7261-7275. doi: 10.1093/nar/gkx438.
Mcm10 is an essential eukaryotic factor required for DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7 and GINS (CMG). DDK is an S-phase-specific kinase required for replication initiation, and the DNA primase-polymerase in eukaryotes is pol α. Mcm10 forms oligomers in vitro, mediated by the coiled-coil domain at the N-terminal region of the protein. We characterized an Mcm10 mutant at the N-terminal Domain (NTD), Mcm10-4A, defective for self-interaction. We found that the Mcm10-4A mutant was defective for stimulating DDK phosphorylation of Mcm2, binding to eighty-nucleotide ssDNA, and recruiting pol α to Mcm2-7 in vitro. Expression of wild-type levels of mcm10-4A resulted in severe growth and DNA replication defects in budding yeast cells, with diminished DDK phosphorylation of Mcm2. We then expressed the mcm10-4A in mcm5-bob1 mutant cells to bypass the defects mediated by diminished stimulation of DDK phosphorylation of Mcm2. Expression of wild-type levels of mcm10-4A in mcm5-bob1 mutant cells resulted in severe growth and DNA replication defects, along with diminished RPA signal at replication origins. We also detected diminished GINS and pol-α recruitment to the Mcm2-7 complex. We conclude that an intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly, and the recruitment of pol α to Mcm2-7.
Mcm10是DNA复制所必需的一种重要真核因子。复制叉解旋酶由Cdc45、Mcm2 - 7和GINS（CMG）组成。DDK是复制起始所需的S期特异性激酶，真核生物中的DNA引发酶 - 聚合酶是polα。Mcm10在体外形成寡聚体，由该蛋白质N端区域的卷曲螺旋结构域介导。我们对位于N端结构域（NTD）的Mcm10突变体Mcm10 - 4A进行了表征，该突变体在自我相互作用方面存在缺陷。我们发现Mcm10 - 4A突变体在体外刺激Mcm2的DDK磷酸化、与80个核苷酸的单链DNA结合以及将polα募集到Mcm2 - 7方面存在缺陷。野生型水平的mcm10 - 4A表达导致芽殖酵母细胞出现严重的生长和DNA复制缺陷，同时Mcm2的DDK磷酸化减少。然后我们在mcm5 - bob1突变体细胞中表达mcm10 - 4A，以绕过由Mcm2的DDK磷酸化刺激减少所介导的缺陷。在mcm5 - bob1突变体细胞中表达野生型水平的mcm10 - 4A导致严重的生长和DNA复制缺陷，同时复制起点处的RPA信号减少。我们还检测到GINS和pol - α募集到Mcm2 - 7复合物的情况减少。我们得出结论，完整的Mcm10卷曲螺旋相互作用表面对于起点解链、解旋酶组装以及将polα募集到Mcm2 - 7至关重要。
