Alver Robert C, Zhang Tianji, Josephrajan Ajeetha, Fultz Brandy L, Hendrix Chance J, Das-Bradoo Sapna, Bielinsky Anja-Katrin
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Natural Sciences, Northeastern State University, 3100 East New Orleans Street, Broken Arrow, OK 74012, USA.
Nucleic Acids Res. 2014 Jul;42(13):8389-404. doi: 10.1093/nar/gku479. Epub 2014 Jun 27.
Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.
基因组的精确复制需要进化上保守的微小染色体维持蛋白Mcm10。尽管Mcm10在DNA复制中的确切作用细节仍存在争议，但它与Mcm2 - 7核心解旋酶、后随链聚合酶、DNA聚合酶α以及复制钳增殖细胞核抗原相互作用。Mcm10缺失导致这些相互作用丧失，进而导致染色体断裂和细胞周期检查点激活。然而，Mcm10在预防DNA损伤方面是否具有积极作用尚不清楚。在此，我们提供的数据确立了Mcm10 N端在抵抗DNA损伤中的新作用。我们发现，在紫外线照射或核苷酸短缺引发复制应激时，Mcm10与9 - 1 - 1钳的Mec3亚基相互作用。我们绘制了Mcm10 N端区域内与Mec3的相互作用结构域，并证明其截短会导致对紫外线敏感。MEC3缺失不会进一步增强这种敏感性，这表明MCM10和MEC3在同一途径中发挥作用。由于在N端截短的mcm10突变体中，紫外线诱导的Rad53磷酸化似乎正常，我们提出Mcm10可能在复制叉重启或DNA修复中发挥作用。
