The Function And Mechanism Of Ubiquitin E3 Ligase Bre1 In Preserving Genome Stability

Maintenance of genome stability is essential to the normal life of cells.It has been shown that the mono-ubiquitylation of histone H2B(ub H2B)is important for proper DNA replication,repair and maintenance of genomic stability.In budding yeast,H2Bub is catalyzed by the ubiquitin E3 ligase Bre1 in cooperation with the E2 ubiquitin conjugating enzyme Rad6.However,how the Bre1-H2Bub pathway is evoked in response to DNA replication or repair remains unknown.In parallel,whether Bre1 has H2Bub-independent functions in the DNA damage response and repair remains to be investigated.To address these questions,we employed a combination of genetic,biochemical and cell biology approaches,and obtained the following results:(1)We performed Bre1-3×FLAG immunoprecipitation and analyzed the associating proteins by mass spectrometry.We found that Rfa1,the large subunit of the ss DNA binding factor RPA,physically interacts with Bre1 in vitro and in vivo,as revealed by coimmunoprecipitation(Co-IP)and GST pull down assays.This interaction is increased in the S/G2 phases or upon DNA damage.Furthermore,we defined the Bre1 residues(L516,L518,D520,D522,L524,L525)and the Rfa1 residue(D465)that are essential to mediate the Bre1-RPA interaction.Mutation of either the residues in Bre1(bre1-6A)or the residue in Rfa1(rfa-D465A)is sufficient to disrupt the interaction in vitro and in vivo.(2)Bre1-mediated ub H2B is essential for proper DNA replication.First,we examined whether the recruitment of RPA or Bre1 at replication forks depends on their interaction.We observed that RPA loading did not decrease in the bre1-6A and rfa1-D465 A mutant cells at replication origins.However,the enrichment of Bre1 and Bre1-mediated ub H2B at replication forks was significantly impaired in bre1-6A or rfa1-D465 A mutation cells,indicating that RPA-Bre1 interaction is required for the recruitment of Bre1 at replication forks.Consequently,disruption of the interaction results in delay of cell cycle progression,impaired DNA synthesis and response to the replication stress,and increased spontaneous DNA damage.Similarly,we found that RPA recruitment at double-stranded break(DSB)ends remains unaffected in the bre1-6A or rfa1-D465 A mutant cells as compared to the WT cells.However,the enrichment of Bre1 or DSBinduced ub H2B was significantly reduced at DSB ends in these mutant cells.As a result,Rad51 loading and DSB repair by homologous recombination(HR)was impaired in rfa1-D465 A or bre1-6A mutant cells.Therefore,the function of Bre1-mediated ub H2B in DNA replication and HR repair depends on the RPA-Bre1 interaction.Thus,we reveal that RPA-mediated recruitment of Bre1 couples ub H2B to DNA replication and repair.(3)Sequence alignment showed that the regions that mediate the RPA-Bre1 interaction are conserved in Bre1 or RPA orthologs.Indeed,we detected strong interaction between human RNF20 and RPA70 by GST pull down assays.Importantly,mutation of the equivalent residues in RNF20(L805,E807,E809,L811 and L812)or that in RPA70(D460)abolished the interaction between RNF20 and RPA70.This result establishes a foundation for testing whether RPA is required to recruit RNF20 to replication forks or DSBs in human cells.(4)By Co-IP assay and GST pull down assays,we found that Bre1 directly interacts with the recombinase Rad51 and that the residues E500 and K502 in Bre1 mediates the Bre1-Rad51 interaction.bre1-EK mutation led to reduced resistances to DNA damaging agents and decreased the DSB repair efficiency,with only ～60% of bre1-EK cells survived.We noted that the enrichment of bre1-EK protein or ub H2B at DSB ends is completely normal in bre1-EK cells.Using in vitro ubiquitination assay,we showed that bre1-EK mutation does not alter the E3 ligase activity of Bre1.(5)Importantly,we demonstrated that Bre1-Rad51 interaction stimulates the replacement of ss DNA-bound RPA by Rad51,stabilizes the Rad51-ss DNA filament,and promotes Rad51-mediated homologous pairing and strand exchange reaction.Interestingly,Bre1 can also suppress Rad51 binding to ds DNA.Finally,we showed that Bre1 antagonizes Srs2 translocating on ss DNA via their direct physical interaction and protects Rad51-ss DNA filament stability from Srs2 dismantling activity.In conclusion,this study revealed the mechanism of how ub H2B responds to DNA replication and repair,and unraveled a recombination mediator activity of Bre1.These results could provide insights into our understanding of the roles of RNF20 and ub H2B in human cells.