| BackgroundIt has been estimated that up to 105 of DNA damage events occur per cell per day and if not be repaired timely and accurately,thoses DNA damage will turn out to be DNA mutations,and eventually,accumulated mutations will cause cell death or even organism death by means of developing fatal diseases such as cancer.Thus,our cells are equipped with several of dedicated DNA damage repair pathways to encounter such huge number and diversity of DNA damage.Amoung all kinds of DNA damage,DNA double strand break(DSB)is the most severe one that in some conditions,a single DSB could causes cell death,and therefore repairing it is of primary important.Following DSB,the activity of PARP1~3(PARPs)is boosted and carry out fast and extensive posttranslational modification(PTM)named poly(ADPribosyl)ation(PARylation)in the vicinity of DSB sites by synthesis covalently linked liner or branched poly ADP ribose(PAR)mainly on PARPs them selves and histones as well as numbers of other proteins.Of note,there are at least three proteins exist in human cell that mediated degradation of PAR,and also the DSB induced PARylation undergoes negative feedback regulation,thus the PARylation is a highly dynamic and transient process.However,PARylation affects DSB repair in the following ways: First,the huge amount of negatively charged PAR molecule covalently linked on chromatin proteins would immediately relax the local chromatin conformation and thus promotes accessibility of DNA damage to repair machinery;Second,PARylation on chromatin proteins will establish repressive chromatin,which would avoid the collision between DNA repair and other processes such as transcription and replication;Third,PARylation,as a PTM,could regulates the target proteins' function by either altering their activity or mediating their association with other proteins;Forth,PAR serve as a fast and strong signal to recruit numbers of proteins which contain several types of PAR-binding protein modules,to initiate DNA repair processes.There are two major repair pathways responsible for DSB repair.The error-prone NHEJ(nonhomologous end-joining)pathway first briefly prosess those two DNA ends at the DSB site to be ligatable and then ligate DSB ends together to recover the DNA molecule integrity.The error-free HR(homologous recombination)pathway utilizes the homologous sequence from the sister chromatin as a repair template to recover both DNA integrity and genetic information integrity.The first step for the HR pathway is to generate a long 3?single strand DNA,by which,the homologous recombination could be fulfilled under the help of RAD51 recombinase.To generate the long 3?single strand DNA,a ‘two step' model has been proposed in which the first step carried out by the endo and exo nuclease activity of MER11 and CtIP respectively generates a short 3?single strand DNA.This is followed by a extend 5? to 3? exo-resection executed by either DNA2 endonuclease or EXO1 exonuclease.EXO1(exonuclease 1)is a member of RAD2 nuclease family which including FEN1,XPG and GEN1.EXO1 possesses 5? to 3? exonuclease activity on double strand DNA and falp structure specific endonuclease activity.Accordingly,EXO1 has been proved to involoved in multiple DNA processes including DNA replication,physiological DNA recombination,DNA repair and activation of cell cycle checkpoint.Knock out of EXO1 casues defects in DNA repair,miosis and elevated susceptibility to some cancer.ObjectivesThe primary objective of this research is to determine new events in DSB repair pathway that be regulated by PARylation and thus help us understanding more thoughly on the role of PARylation in DSB repair.Specifically,we focus on on the phenomenon that EXO1 is rapidly relocated to DNA damage site,and tring to determine the underling mechanism of the EXO1 fast relocation upon DSB and its contribution to HR pathway.Methods and resultsFisrt,by ultizing laser microirradiation and live cell image,we monitored the dynamics of the GFP-EXO1 relocation upon DNA damage under various conditions.We find that GFP-EXO1 is rapidly recruited to DNA damage and its dynamics resemble the dynamics of DNA damage induced PARylation.By interfering the DSB induced PARylation,we demonstrated that the fast recruitment of EXO1 to DSB is dependent on PARylation.Further dissection of the protein segments revealed that the PIN domain mediates the fast recruitment of EXO1,suggestes that the EXO1 PIN domain directly binding with PAR.Second,we tested the hypothesis that the PIN domine of EXO1 directly recognizes PAR.To do this,reciprocal pull-down assy between purified EXO1 fragment recombinate proteins and in vitro synthesized PAR revealed their direct binding in vitro.The reciprocal Co-IP between endogeneous EXO1 and DNA damage induced PAR further supports their direct binding in vivo.We also explored other PIN domains on their PAR binding ability,as revealed by in vitro assay,we found two other PIN domains could recognize PAR and this was further supported by in vivo experiments.Taken together,we find that the PIN domain as a new module for PAR binding is responsible for the fast recruitment of EXO1 to DNA damge site.Third,we further analysed the functional importance of PARylation mediated fast recruitment of EXO1 to DNA damage repair.By screening several natural variants which harbor single amino acid substitution within EXO1 PIN domain,we find a varient R93 G which abolishes the EXO1 PAR-binding ability yet keeps the intact nuclease activity.This varient serves as an ideal model to dissect the ability of PAR binding from nuclease activity,as these two function are carried out by the same domain potentially prevents us to do any deletion to separate those functions.By knock down the endogeneous EXO1 and rescued with either wild type EXO1 or R93 G varient,we found that abolishing the EXO1 fast recruitment largely delayed the formation of single strand DNA in damage site as revealed by RPA immunostaining.A homologous repair assy further demostrated that delayed EXO1 recruitment causes moderate reduction of homologous recombination ratio.Thus we proved that the PAR dependent recruitment of EXO1 promotes DSB HR repair poteintially by interfering the pathway choice step.Lastly,to further understanding the complexity of EXO1 regulation,we performed large scale tandem affinity purification of EXO1 associated proteins and follow by mass spectrum.We recovered a larged number of previously reported EXO1 interacting proteins which suggests the high performance of our tandem affinity purifiniton process.By comparing the EXO1 interactome with PARylated proteome reported by two indpendent groups,we find limited number of proteins that both associated with EXO1 and be PARylated after DNA damage.However,as those proteins has relatively high incidence to associate with EXO1 and also be PARylated,the results still suggestive.We also find a batch of new EXO1 associated protein,amoung which,we selected USP7 for functional study.Our preliminary data suggest that USP7 stabilizes EXO1 protein by deubiquitination,however,more experiments are needed to fully understand this hypothesis.ConclusionsUpon all the results above,we would like to draw our conlusions as below,1.The fast recruitment of EXO1 to DSB site promotes HR repair;2.PIN domain is a new module that recognizes PAR;3.The PAR binding activity of PIN domain is independent of its nuclease activity. |
PDF Full Text Request