Molecular Mechanism Of Replication Fork Reversal In Higher Eukaryotes

The faithful and complete replication of DNA is a key event essential for the maintenance of genome stability.However,DNA replication is frequently challenged by both endogenous and exogenous stresses,which stalls replication forks.Fork stalling is a main resource of fork collapse and genome instability.In higher eukaryotes,replication fork reversal is a global and regulated response to different sources of replication stress with both physiological and pathological outcomes.Previous studies have demonstrated that fork reversal is generally catalyzed by SNF2-family DNA translocases including HLTF,ZRANB3 and SMARCAL1.The displacement and annealing of the nascent and parental DNA strands catalyzed by these enzymes during fork reversal generates positive super-helical strain in the newly replicated sister chromatids behind the forks,which prevents further fork reversal.Meanwhile,the accumulation of extensive topological barriers is also a threat of genome instability.The resulting topological barriers thus must be relieved by topoisomerases for reversal to proceed efficiently.However,which topoisomerases are responsible for removing super-helical strain in the newly replicated sister chromatids remains largely unknown,as is how the overall extent of fork reversal is controlled in cells,and how topoisomerases coordinate with known fork-remodeling factors or other enzymes to promote extensive fork reversal.In this study,using proximity ligation assay(PLA)and isolation of proteins on nascent DNA(i Pond),we present evidence that topoisomerase ??(TOP2A)accumulates at stalled replication forks in a manner dependent on SNF2-family DNA translocases HLTF,ZRANB3 and SMARCAL1.In addition,TOP2 A depletion decreases the regressed arm length at reversed replication forks,as well as the fork reversal frequency,suggesting that TOP2 A may promote the extension of fork reversal,thereby stabilizing reversed forks.Notably,we demonstrate that SUMOylation of TOP2 A is greatly enhanced upon replication stress,and further reveal that the SUMO E3 ligase ZNF451 is responsible for TOP2 A SUMOylation.Interestingly,we find SUMOylated TOP2 A is able to recruit the SUMO-targeted DNA translocase PICH to stalled replication forks.In vitro assays show that PICH branch-migrates four-way junction structures,but fails to catalyze regression of model replication forks.Similar to TOP2 A,downregulation of ZNF451 or PICH decreases the regressed arm length of the reversed fork and the fork reversal frequency as well.In line with this,disruption of ZNF451-TOP2A-PICH axis renders cells more sensitive to replication stress.Based on these findings,we propose a two-step model for fork reversal.In the first step,DNA translocases HLTF,ZRANB3 and SMARCAL1 initiate fork reversal,creating super-helical strain in the newly replicated sister chromatids.In the second step,TOP2 A accumulates at stalled replication forks,resolves topological barriers generated by the first step,and drives extensive fork reversal by recruiting SUMO-targeted translocase PICH to stalled forks.