Screening Suppressors Of Atr Mutant In Arabidopsis By Activation Tagging

The ultimate goal of all organisms is to pass the genetic information(DNA)to the next generation.However DNA is constantly threatened by all kinds of damage.To maintain genome stability,organisms have evolved elaborate mechanisms to repair DNA damage.In animal DNA damage repair system,the protein kinases ATM and ATR are the two most important proteins,which phosphorylate their downstream protein kinases CHK1 and CHK2,respectively,and thereby further regulate other DNA damage repair genes.In plants,no CHK1 homolog has been identified.Therefore,it remains elusive how ATR regulates DNA damage repair in plants.The major goal of this study is to identify key genes in plant ATR pathway,and to elucidate its mechanism.Arabidopsis atr mutant is very sensitive to DNA damage agent hydroxyurea(HU).Based on the phenotype,this study aims to screen suppressors of atr mutant by activation tagging.We successfully identified a suppressor named 2-12.Through inverse-PCR analysis,we found that the activation tag was inserted inside the PRL1 gene.In order to further confirm that prl1 is a suppressor of atr,we crossed atr with mcr1,another prl1 T-DNA insertion mutant.We found that the phenotype of atr mcr1 double mutant is consistent with 2-12,indicating that mutation of PRL1 gene did suppress the phenotype of atr.PRL1,MAC3,CDC5 and MOS4 form MOS4 complex,which is involved in RNA splicing.WEE1 and SOG1 are two known downstream genes of ATR.In order to study the relationships between these genes and PRL1,we tested their interactions using yeast two-hybrid assays and split luciferase assays.We found that PRL1 did not interact with ATR,WEE1 or SOG1.But ATR interacted with MAC3 A,MAC3B and MOS4;WEE1 interacted with MAC3 A,MAC3B and CDC5;SOG1 weakly interacted with MAC3 B and MOS4.PRL1 is also a subunit of CUL4-based E3 ligase complex,which regulates protein degradation.In animals,ATR regulates DNA replication stress responses by positively regulating replication protein A(RPA)complex.We hypothesize that PRL1 may negatively regulate DNA replication stress responses by degrading RPA.We found that PRL1 did interact with RPA2 and RPA3,and the interactions were enhanced by HU treatment.In the in vitro degradation assay,PRL1 did not affect the degradation of RPA2 A but promoted the degradation of RPA3 A.Our study found that PRL1 is a key negative regulator downstream of ATR,providing new clues to further study the mechanism of ATR.Based on these results,we propose the following model: In response to DNA replication stress,ATR negatively regulates PRL1 directly or indirectly,and thus inhibits PRL1-mediated RPA degradation,which results in increased RPA level and promotes DNA repair.PRL1 may also negatively regulate DNA replication stress responses by regulating alternative splicing of some key genes.The mechanism of PRL1 needs to be further studied.