RNF4 Controls The Extent Of Replication Fork Reversal To Preserve Genome Stability

Accurate duplication of chromosomal DNA during the S phase of cell cycle is a prerequisite for faithful transmission of genetic material from parent to offspring.Various replication stresses,originating from endogenous or exogenous can hinder replication fork progression.Timely restart of stalled replication forks is vital for maintenance of fork integrality,genome stability and genetic information accuracy.In higher eukaryotes,replication fork reversal is a sophisticated and tightly regulated cellular response to all kinds of replication stress,in which the two extruded nascent DNA strands anneal to form a Holliday junction like structure.This transient remodeling of replication forks can actively restrict DNA synthesis under suboptimal condition,prevent accumulation of ss DNA,and unwound to assist completion of replication in appropriate circumstance.This dynamic reversible process efficiently stabilizes stalled replication forks.Previous studies have shown that recombinase RAD51,cooperate with translocases SMARCAL1、ZRANB3 and HLTF to promote fork reversal.But the exact mechanisms under this is elusive.Our studies present that RAD51 recombinase as well as multiple fork remodelers such as SMARCAL1,ZRANB3,and HLTF initiate limited fork reversal,thereby introducing positive supercoils into the newly synthesized chromatids to prevent futher reversal of stalled forks.Then topoisomerase TOP2 A recognizes and removes the topological stress.As the same time,TOP2 A is SUMOylated by SUMO E3 ligase ZATT,translocase PICH targets SUMOylated TOP2 A to accumulate at stalled forks and then drives extensive fork reversal.Thus we firstly identify that fork reverse carried out via a two-step process: firstly,RAD51 together with SMARCAL1、ZRANB3、HLTF and other proteins to initiate limited fork reversal,introducing positive supercoils in the nascent sister chromatids.Secondly,TOP2 A senses and releases the topological stress,meanwhile SUMOylated TOP2 A recruits PICH to drive extensive fork reversal.Extensive fork reversal protects stalled forks from unscheduled restart,thus restraining forks collapse.However,uncontrolled fork reversal also led to pathological degradation of nascent DNA,which ultimately cause a defective replication restart under proper condition,the exact mechanisms of how cell control the extent of fork reversal remain poorly understood.Here we show that SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled fork upon replication arrest and targets 20 lysine residues of SUMOylated TOP2 A for ubiquitination and degradation.Downregulation RNF4 led to hyper-accumulation of TOP2A-ZATT-PICH complex at stalled forks,which in turn resulted in aberrant forks collapse,genome instability.Our studies not only uncover a previous unidentified two-step model of fork reversal but also illustrate the mechanism that cell regulates the extent of fork reversal.These results complete the mechanisms of fork reversal and restart as well as reveal the way of higher eukaryotes faithfully duplicate DNA and transmit genetic information to offspring under replication stress.