期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：3
页码：E249-E258
DOI：10.1073/pnas.1422003112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceCell division control protein 45 (Cdc45), a RecJ homologue, is essential in all eukaryotes. Cdc45 functions with the replisome CMG helicase where minichromosome maintenance (Mcm2-7) proteins provide motor activity for unwinding duplex during replication. We report that the dynamic gate between Mcm subunits 2 and 5, which is essential for the initial loading of the motor, may be an Achilles heel because the leading strand may slip from its central channel in an open gate state. Studies show that the side channel formed by the Cdc45 and GINS works as a trap and guards this gate; the Recombination protein J fold is key for this activity. We propose that this new function for Cdc45 will be important for fork integrity during the S-phase in response to double-strand breaks or replication stress. DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.
