Study On Mutants Of Key Proteins FEN1 And Pol?arginine Methylation Sites In The Base Excision Repair Pathway

DNA is the carrier of life genetic information.The Integrity and Stability of DNA is extremely important for maintaining normal life.DNA repair pathway is essential for genome integrity and stability.Functional disorders of DNA repair pathway,especially base excision repair(BER)pathway,will lead to serious diseases,including cancer.Based on how many nucleotides involved in repair,BER can be divided into two types:short patch BER and long patch BER.Both are initiated by glycosylase removing the damaged base and followed by APE1 nicking the DNA backbone,Polp filling in the gap and ligase sealing the nick as in SP-BER.In LP-BER,FEN1 is needed to cleave the flap structure caused by Polp and then the nick is sealed by ligase.Previous researches show that there is more than 105 BER events happened in each mammalian cell daily.Such a huge repair events require rigorous and precise control of every process in the BER pathway in a temporal and spatial manner.However,we know little about the molecular mechanisms of this regulation.In our previous work,we found that several key BER enzymes are regulated by arginine methylation.For example,the recruitment of FEN1 is regulated by arginine methylation as well as the detachment from the damaged sites of DNA.The activity of Polp and its interaction with PCNA are also regulated by arginine methylation.Therefore,we hypothesized that mutation at arginine methylation site leads to lower efficiency in DNA damage repair and thus results in tumors and other diseases.To verify this hypothesis,we studied the arginine methylation mutants of FEN1 and Pol?,trying to explore the correlation between regulation and tumorigenesis.FEN1 is a structure specific endonuclease.It is the downstream enzyme of Pol?which is responsible for the removal of Flap structure in LP-BER repair.FEN1 is recruited to the DNA damage site by PCNA and the combination and dissociation of FEN1 with PCNA are the key steps for FEN1 to function.Previous studies showed that the phosphorylation of FEN 1 promotes the detachment of FEN1 and PCNA by altering the charge at the surface of FEN1.Since there are a large number of phosphate kinases in cells,we wondered how FEN1 could bind to PCNA normally.Finally,we found that arginine methylation of FEN1 is the answer.With the help of PRMT5,it is arginine methylation at the number 192 site of FEN1,termed symmetric dimethylarginine(SDMA),that inhibits phosphorylation of FEN41 and ensures the combination with PCNA.This confirmed the important role of arginine methylation in the repair of DNA damage.In addition to the SDMA 192 site of FEN1,we recently found a new FEN1 arginine methylation site,R378.This site is different from R192,which is catalyzed by PRMT1 to produce asymmetric dimethylarginine(ASDMA).These observations suggest that ASDMA of the 378 site may have different functions from SDMA of R192 site,which need further study.As the stability of FEN1 decreased after FEN1 R378 mutation,we hypothesized that the cells containing FEN1 R378 mutation would be more sensitive to DNA damage,and the population of mutants would be more likely implicated in tumorigenesis..Thus,we tried to find the FEN1 R378 mutation in the tumor samples,but failed.It might be due to the important role it played.It has been established that Pol? functions as a key enzyme in BER pathway,which function is also regulated by arginine methylation.DNA Pol? mutations have been detected in various types of cancers,implying a link between mutations and carcinogenesis.However,it is still unclear whether and how these mutations of DNA Polp lead to cancer onset and progress.We studied R152C Pol?,a mutation of arginine methylation site found from eolon cancer samples to explore the relevance between posttranslational modification dysregulation and pathological processes.In the current work,we showed that R152C decreases DNA Polp polymerase activity and impairs BER efficiency.Cells harboring the R152C mutation of DNA Pol? are more sensitive to DNA damaging reagents such as methyl metihanesulfonate(MMS)and H2O2 compared with wild type(WT)cells.Moreover,the R152C containing cells display a higher percentage of chromatid breakages and aneuploidy and form foci more easily than the WT cells.Taken together,our data indicate that the R152C mutation has the potential to drive tumorigenesis.R152C Pol? found in tumor samples is somatic mutation and can not be inherited So we searched out another inheritable arginine methylation mutant,R137Q Pol?,and constructed a mouse model to explore the significant influence caused by arginine methylation deletion at the living onanism level.R137Q Pol? is identified as another arginine methylation mutant in tumors which is also an SNP site,occurring in somatic cells and germline cells,as the most common genetic variant.Previous studies at the molecular level demonstrated that mutations in R137 resulted in a decrease in Pol? polymerase activity and a reduction in efficiency of DNA repair.Pol? R137Q homozygous knock-in mouse embryos were typically smaller in size and had a higher mortality rate(21%).These embryonic abnormalities were caused by decreased cell proliferation and increased apoptosis.In R137Q knock-in mouse embryos,the BER efficiency was severely impaired,which subsequently resulted in double-strand breaks(DSBs)accumulation and chromosomal aberrations.In summary,we chose arginine methylation mutants of key BER enzymes:R378K FEN1,R152C Polp and R137Q Pol? as the research subjects,to provide evidence for protein arginine methylation,an important posttranslational modification,which plays an important role in regulating the efficiency of BER pathway by controlling stability or activity of proteins at molecular level,cell level and animal level.The mutation of arginine methylation sites causes the dysfunction of BER pathway,resulting DNA metabolism disorder,genomic instability in cells,which will lead to the physiological disorders and diseases,including tumor.Our work is a completion of regulation mechanism of base excision repair pathway,providing a theoretical basis for the diagnosis and treatment of diseases related to DNA repair.It can be used as a drug target and clinical diagnostic marker in individual medicine.