The Role And Mechanism Of Bre1- Dependent H2b Ubiquitination In DNA Repair By Homologous Recombination

DNA double-strand breaks(DSBs)are one of the most deleterious DNA lesions.Inaccurate repair of DSBs can cause chromosomal deletions,insertions,rearrangement and translocation.These events can result in genome instability and eventually induce mutagenesis or cell death.DSBs are repaired by two major pathways that conserved from yeast to higher eukaryotes.One is called non-homologous end joining(NHEJ)and the other is homologous recombination(HR).It has been well established that chromatin status and histone modification play important roles in DNA doublestrand repair.However,a full understanding of their functions and mechanisms in DSB repair remains incomplete.Histone H2B is monoubiquitylated on K123 by Bre1,an E3 ubiquitin ligase with RING domain,with E2 ubiquitin binding enzyme Rad6 in Saccharomyces cerevisiae.It has shown that Bre1-dependent H2 B ubiquitination plays an important role in many DNA transactions including replication,transcription,meiosis,centromere stability and telomere terminal processing.Previous study showed that bre1 ? mutant is sensitive to ionizing radiation,suggesting a role of Bre1 in DNA damage repair.In this study,we explored the function and molecular mechanism of Bre1-dependent H2 B ubiquitination in the homologous recombination pathway of DSBs repair and obtained the following results:1)Compared with wild-type cells,bre1? mutation is susceptible to DNA damage drugs by transient or continuous treatment,and survival rate significantly reduced.It has found that the BRE1 knockout led to a significant decrease in the efficiency recombination repair by ectopic homologous recombination experiment and gene targeting experiment.Moreover,Bre1 promotes HR repair dependently on H2 B ubiquitination.Accordingly,ChIP-qPCR results showed that Bre1 protein was directly recruited to broken end,which stimulates local H2 B ubiquitination.2).Southern blot analysis showed that Bre1 inhibits end processing by both Sgs1-Dna2 and Exo1 pathways.This is because of that deletion of BRE1 abolished H3K79 methylation and subsequent recruitment of Rad9,a known negative regulator of resection,resulting in accelerated resection.Nonetheless,the HR repair defect is not due to the fast resection in bre1 ? mutant.3)Further,we revealed that the recruitment of ssDNA binding protein RPA complex and recombinase Rad51 are decreased significantly at DSB end by ChIPqPCR,no matter in bre1 deletion mutant or in H2B-K123 ubiquitination defective mutant.Consistently,Chromatin fraction experiments showed Rad51 associated with chromatin significantly decreased,whereas Rad51 in solution fraction significantly increases upon DNA damage.Furthermore,we found that the HR defect correlates with impaired histone loss in bre1? cells.Therefore,we reasoned that bre1-dependent H2 B ubiquitination may affect the recruitment of repair proteins Rad51 by promoting the removal of histones.4)To test the above hypothesis,we overexpressed RAD51 in bre1?mutant.Rad51 overexpression can competitively inhibit histone H3 retention at DSB ends.Accordingly excessive Rad51 can partially rescue the HR repair defects in bre1? mutant.In addition,We found that deletion of CAC1 significantly rescues the defect HR repair in bre1? mutant cell.which further strengthens the notion that Bre1 play an important function in histone removal.5)We also explored the possible mechanism of Bre1 promoting histone removal.We found that the role of Bre1 in promoting histone removal is irrelevant to its function in suppressing end resection.On the other hand,we showed that the role of Bre1 in histone removal is likely independent of the chromatin remodeler complexes INO80,SWR,RSC and Fun30 since these remodelers are properly recruited to DSBs ends in bre1 ? cells.Altogether,our results suggest a model that Bre1-mediated H2 B ubiquitination promotes HR via regulating histone dynamics at DSB ends.