Molecular Mechanism Of The ZATT-TOP2A-PICH Axis In Replication Stress Response

Faithful and complete replication of genomic DNA requires a variety of protective mechanisms to respond to both endogenous and exogenous replication stress.In higher eukaryotes,replication fork reversal is a common and tightly regulated cellular response to replication stress.It is defined as the transformation of a typical replication fork from a three-way junction into a Holliday junction(HJ)structure.Interestingly,we find that the maximum length of the regressed arms measured by EM could be up to several kilobases,revealing the occurrence of the extensive replication fork reversal.The displacement and annealing of the nascent and parental DNA strands will generates topological barriers in the newly replicated sister chromatids behind the forks,which prevents further regression of stalled replication forks.Thus,the resulting topological barriers must be relieved by topoisomerases for extensive reversal to proceed efficiently.However,which and how topoisomerases are responsible for resolving topological barriers during replication fork reversal remains unknown.In this study,TOP2 A is proved to be the main topoisomerase that resolves the topological barriers in the newly replicated sister chromatids during fork reversal.We show that,upon replication stress,the SUMO E3 ligase ZATT facilitates TOP2 A SUMOylation at lysines 1228 and1240,thereby recruiting SUMO-targeted DNA translocase PICH to stalled replication forks.Both the DNA translocase activity of PICH and its ability to interact with SUMOylated TOP2 A are critical for its function in fork reversal.SNF2-family DNA translocases HLTF,ZRANB3,and SMARCAL1 are known to be required for initiating replication fork reversal upon replication stress.Here,we show that HLTF,ZRANB3,and SMARCAL1 are required for the ZATT-TOP2A-PICH axis recruitment to stalled replication forks.Surprisingly,PICH is unable to catalyze the initiation step of replication fork reversal but can branch migrate four-way junctions to promote extensive replication fork reversal in vitro.Taken together,these results suggest that upon replication stress,fork reversal occurs via a sequential two-step process:(1)HLTF,ZRANB3,SMARCAL1,and/or other fork remodeling factors converts model replication fork into four-way junction structures to initiate limited fork reversal,and(2)the ZATT-TOP2A-PICH axis branch-migrates the resulting four-way junctions to drive extensive fork reversal.