You Zhiying, Masai Hisao
Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-8506, Japan.
Nucleic Acids Res. 2017 Jun 20;45(11):6494-6506. doi: 10.1093/nar/gkx269.
Mini-chromosome maintenance (Mcm) is a central component for DNA unwinding reaction during eukaryotic DNA replication. Mcm2∼7, each containing a conserved ATPase motif, form a six subunit-heterohexamer. Although the reconstituted Mcm2∼7-Cdc45-GINS (CMG) complex displays DNA unwinding activity, the Mcm2∼7 complex does not generally exhibit helicase activity under a normal assay condition. We detected a strong DNA strand annealing activity in the purified mouse Mcm2∼7 heterohexamer, which promotes rapid reassociation of displaced complementary single-stranded DNAs, suggesting a potential cause for its inability to exhibit DNA helicase activity. Indeed, DNA unwinding activity of Mcm2∼7 could be detected in the presence of a single-stranded DNA that is complementary to the displaced strand, which would prevent its reannealing to the template. ATPase-deficient mutations in Mcm2, 4, 5 and 6 subunits inactivated the annealing activity, while those in Mcm2 and 5 subunits alone did not. The annealing activity of Mcm2∼7 does not require Mg2+ and ATP, and is adversely inhibited by the presence of high concentration of Mg2+ and ATP while activated by similar concentrations of ADP. Our findings show that the DNA helicase activity of Mcm2∼7 may be masked by its unexpectedly strong annealing activity, and suggest potential physiological roles of strand annealing activity of Mcm during replication stress responses.
微型染色体维持蛋白（Mcm）是真核生物DNA复制过程中DNA解旋反应的核心组成部分。Mcm2∼7各包含一个保守的ATP酶基序，形成一个六亚基异源六聚体。尽管重组的Mcm2∼7-Cdc45-GINS（CMG）复合物具有DNA解旋活性，但在正常检测条件下，Mcm2∼7复合物通常不表现出解旋酶活性。我们在纯化的小鼠Mcm2∼7异源六聚体中检测到很强的DNA链退火活性，该活性促进被置换的互补单链DNA快速重新结合，这表明其无法表现出DNA解旋酶活性的一个潜在原因。实际上，在存在与被置换链互补的单链DNA时，可以检测到Mcm2∼7的DNA解旋活性，这将阻止其与模板重新退火。Mcm2、4、5和6亚基中的ATP酶缺陷突变使退火活性失活，而仅Mcm2和5亚基中的突变则不会。Mcm2∼7的退火活性不需要Mg2+和ATP，高浓度的Mg2+和ATP会对其产生不利抑制，而相似浓度的ADP则会激活它。我们的研究结果表明，Mcm2∼7的DNA解旋酶活性可能被其意外强大的退火活性所掩盖，并提示了Mcm链退火活性在复制应激反应中的潜在生理作用。
