Regulation of CMG Helicase Disassembly During Replication Termination

Abstract

During DNA replication, the replicative helicase, CMG, unwinds the parental duplex at the leading edge of each replication fork. CMG unwinds DNA by encircling the leading strand template and excluding the lagging strand template, which interacts with the surface of the helicase. When two forks meet during replication termination, the last parental duplex is unwound, the leading and lagging strands of the converging forks are ligated, and the CMG helicase is unloaded from chromatin. CMG unloading occurs only after the leading strand of one fork is ligated to the lagging strand of the converging fork, indicating that double-stranded DNA (dsDNA) enters CMG’s central channel before helicase unloading. In yeast, CMG unloading involves the SCFDia2 ubiquitin ligase, which mediates formation of K48-linked ubiquitin chains on CMG's Mcm7 subunit. The ubiquitin chains are recognized by the p97Ufd1/Npl4 ATPase complex, which extracts the helicase from chromatin. This dissertation addresses the mechanism and regulation of CMG unloading in higher eukaryotes. We show that in Xenopus egg extracts, Mcm7 is ubiquitylated by the CRL2Lrr1 ubiquitin ligase. CRL2Lrr1 is recruited when two replication forks converge during termination, ensuring that CMG is not unloaded prematurely. Furthermore, unloading of CMG is coordinated with the removal of CMG-associated replication fork proteins. We further addressed how CRL2Lrr1 recruitment is coupled to replication termination. We show that CMG unloading does not require encounter of the helicase with dsDNA, nor does it depend on the collision of two forks, arguing that other signals for CRL2Lrr1 recruitment must exist. Indeed, our evidence suggests that CRL2Lrr1 recruitment depends on loss of an interaction between the lagging strand template and the exterior of CMG. Finally, we show that the replication protein, Mcm10, and a termination-specific factor, And-1, regulate Mcm7 ubiquitylation. In summary, we have identified a set of proteins that contribute to CMG unloading and characterized the signals that trigger replisome disassembly.