Abstract
SummaryThe convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.
Highlights
In contrast to the initiation and elongation stages of eukaryotic DNA replication, which have been studied intensively over recent decades (Bell and Labib, 2016; Burgers and Kunkel, 2017; Deegan and Diffley, 2016), very little is known about the process of DNA replication termination, which occurs whenever two replisomes from neighboring replication origins meet each other (Dewar and Walter, 2017)
The final stages of DNA synthesis involve at least five processes that are particular to termination (Dewar and Walter, 2017): the convergence and encounter of the two replisomes, gap filling between the leading strand at one fork and the lagging strand of the other (Dewar et al, 2015), regulated disassembly of the replisome (Dewar et al, 2017; Maric et al, 2014, 2017; Moreno et al, 2014; Sonneville et al, 2017), and decatenation of the sister chromatids that are the products of replication
A ‘‘minimal replisome’’ assembles around CMG at each nascent DNA replication fork, with DNA polymerase a making primers for lagging-strand synthesis, whereas DNA polymerase ε extends the leading strand, and the type II topoisomerase Top2 removes supercoils to allow fork progression. Further development of this reconstituted replication system (Yeeles et al, 2017) added components of the replisome progression complex that assembles around the yeast CMG helicase (Gambus et al, 2006), including the type I topoisomerase Top1, and added DNA polymerase d (Pol d) and other factors that are required for lagging-strand synthesis
Summary
In contrast to the initiation and elongation stages of eukaryotic DNA replication, which have been studied intensively over recent decades (Bell and Labib, 2016; Burgers and Kunkel, 2017; Deegan and Diffley, 2016), very little is known about the process of DNA replication termination, which occurs whenever two replisomes from neighboring replication origins meet each other (Dewar and Walter, 2017). The final stages of DNA synthesis involve at least five processes that are particular to termination (Dewar and Walter, 2017): the convergence and encounter of the two replisomes, gap filling between the leading strand at one fork and the lagging strand of the other (Dewar et al, 2015), regulated disassembly of the replisome (Dewar et al, 2017; Maric et al, 2014, 2017; Moreno et al, 2014; Sonneville et al, 2017), and decatenation of the sister chromatids that are the products of replication. The final stages of DNA unwinding and DNA synthesis are thought to require clockwise rotation of the two converging replication forks to transfer the topological stress behind the two replisomes in the form of intertwines or precatenanes between the replicated sister chromatids (Keszthelyi et al, 2016; Schalbetter et al, 2015)
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