Significance Fork regression is a way of circumventing or dealing with DNA lesions and is important to genome integrity. Fork regression is performed by double-strand DNA ATPases that initially cause… Click to show full abstract
Significance Fork regression is a way of circumventing or dealing with DNA lesions and is important to genome integrity. Fork regression is performed by double-strand DNA ATPases that initially cause newly synthesized strands to unpair from the parental strands, followed by pairing of the new strands and reversal of the fork. This study shows that Mcm10, an essential replication factor, efficiently anneals complementary strands and also inhibits fork regression by SMARCAL1. Moreover, the study localizes the Mcm10 DNA-binding domain to the N-terminal domains of the replicative CMG helicase at the forked nexus. Thus, forks that are unimpeded would contain Mcm10 at a strategic position where its DNA-binding and/or annealing function may block fork regression enzymes and thereby protect active forks from becoming reversed. The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, GINS) tightly binds Mcm10, an essential replication protein in all eukaryotes. Here we show that Mcm10 has a potent strand-annealing activity both alone and in complex with CMG. CMG-Mcm10 unwinds and then reanneals single strands soon after they have been unwound in vitro. Given the DNA damage and replisome instability associated with loss of Mcm10 function, we examined the effect of Mcm10 on fork regression. Fork regression requires the unwinding and pairing of newly synthesized strands, performed by a specialized class of ATP-dependent DNA translocases. We show here that Mcm10 inhibits fork regression by the well-known fork reversal enzyme SMARCAL1. We propose that Mcm10 inhibits the unwinding of nascent strands to prevent fork regression at normal unperturbed replication forks, either by binding the fork junction to form a block to SMARCAL1 or by reannealing unwound nascent strands to their parental template. Analysis of the CMG-Mcm10 complex by cross-linking mass spectrometry reveals Mcm10 interacts with six CMG subunits, with the DNA-binding region of Mcm10 on the N-face of CMG. This position on CMG places Mcm10 at the fork junction, consistent with a role in regulating fork regression.
               
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