Functions Of Drosophila Mre11 Arginines Methylation At DNA Damage Response

MRN?Mre11/Rad50/Nbs1?complex is the major complex at DNA damage response and highly conserved in eukaryotes.MRN complex plays a critical role in multiple processes,such as DNA damage recognition,signal transduction and downstream repair processes.Mre11 protein is a cenral component in MRN compelx.Mre11 is a key player for DNA double strand break repair and has nuclease activities which can resect DNA broken ends.In human,the mutated mre11 gene results in ataxia telangiectasia-like disorder syndrome?ATLD?.When mre11 gene deletion in mouse,the embryonic will lethality.Mre11 is very important for maintaining genomic stability and suppressing telomere fusion and activating G2/M checkpoint after DNA damage.Previous studies showed that post-translational modifications were essential for the functions of DNA binding proteins.Studies using mammalian systems show that Mre11 interact with PRMT1.Mre11 has a glycine-arginine-rich region?GAR domain?in C-terminal which can be arginine-methylated by protein arginine methyltransferase PRMT1.The modification is involved in Mre11 functional regulations during DNA damage repair.For example,mre11RK/RK knock-in mice and MEFs which arginines substituted with lysines have shown that are hypersensitive to gamma ray radiation.The cells display significant post-irradiation defects in G2/M cell cycle checkpoint and ATR/CHK1 signaling activation,increasing in aberrant chromosomes and reducing RPA and RAD51 foci.Drosophila Mre11 is highly conserved and the major defect in mre11 mutants is telomere-telomere associations and G2/M checkpoint defect.While mammalian PRMT1 interact with Mre11 and catalyze its methylation,whether or not Drosophila Mre11 is methylated by DART1,the Drosophila homolog of PRMT1,remains unknown.Therefore,we research functions of Drosophila Mre11 at DNA damage response.We aligned the Mre11 protein sequence from Drosophila and multiple specifies using Clustal W2 software.Alignment shows that Drosophila Mre11 has a GAR motif at its C-terminus and there are six arginines in this GAR region.They are Arg559,Arg 563,Arg 565,Arg 569,Arg 571 and Arg 579.In vitro methylation results show that Mre11 GAR domain is methylated by DART1.Mre11 is detected by arginine methylation antibodies recognizing R559,563,565 and 569 sites.The methylated Mre11 exists as a soluble nucleoplasmic or chromatin-binding protein.we next knock down dart1 expression by siRNA,the result shows that methylation of Mre11 is reduced.We also find Mre11 protein and DART1 are interact with each other through a co-immunoprecipitation?Co-IP?experiment.Mre11 GAR motif is not required for the Drosophila Mre11-DART1 interaction.In order to address these questions and understand the wider functions of arginine methylation of Drosophila Mre11,we construct series of mutation flies.They are mre11 R559A^1-1I1,mre11 R559A^1-1K12,mre11 R559A^1-1T10,mre11 R563A^3-1D5,mre11 R565A^2-7M5,mre11 R569A^6-34H1,mre11 4RA^2-3P3,mre11 4RA^2-3I9.The mutated flies are hypersensitive to ionizing radiation.These data shows that arginine methylation of Drosophila Mre11 play a important role at DNA damage response.And understanding the mechanism is essential for human diseases prevention and treatment.