Biological Effects Of ALKBH2 And ALKBH3 On Replication Of N1-methyladenine In Human Cells

N1-methyladenine(1m A)is an important alkylation modification and widespread in DNA.The presence of 1m A will affect the DNA structure,thereby affecting the efficiency of protein synthesis,and even change the phenotype of organisms.It has been verified that Alk B in Escherichia coli can demethylate 1m A,and the human homologous proteins ALKBH2 and ALKBH3 can also demethylate 1m A in Escherichia coli.However,there are few studies on the repair of 1m A by ALKBH2 and ALKBH3in human cells and the specific repair mechanism is still unclear.In this study,CRISPR-Cas9 gene editing technology was used to construct ALKBH2 or ALKBH3 knock-out cell lines,and then combined shuttle plasmid containing 1m A with next generation sequencing,the biological effects of ALKBH2 or ALKBH3 on replication of 1m A were studied.The main research contents were summarized as follows:(1)Construction and validation of ALKBH2 or ALKBH3 mutated cell lines.Firstly,we constructed pX330A vector containing sgRNA which targeted ALKBH2 or ALKBH3 gene,these vectors were then transfected into HEK 293T cells,the selected monoclonal cell lines were cultured and verified by sequencing,and sequencing results showed that ALKBH2 and ALKBH3 gene were mutated respectively.Finally,protein expression levels in cell lines were verified via Western Blotting.The results showed that we successfully constructed ALKBH2 or ALKBH3 mutated cell lines.(2)Validation of alkylation repair of ALKBH3.Firstly,we isolated the RNA of ALKBH3^-/--12 cell lines and analysed m RNA level with q RT-PCR.Compared to wild-type cells,the relative m RNA level of ALKBH3 gene in ALKBH3^-/--12 cells was not prominently reduced.Then,we extracted and characterized the nuclear protein and cytoplasmic protein of ALKBH3^-/--12 cells by Western Blotting,the nuclear protein of ALKBH3 had no obvious difference compared with the wild type cells.So,we can conclude that the deletion sequence of N-terminal does not influence nuclear localization of ALKBH3.The nuclear localization signal of ALKBH3 need to be further studied.Finally,we verified the repair function of alkylation modification of ALKBH3through cytotoxicity test.(3)Biological effects of ALKBH2 and ALKBH3 on replication of 1m A.Firstly,the plasmids containing 1m A were transfected into ALKBH2 or ALKBH3 mutated cell lines.The replication products were extracted and the parental plasmids were removed.Nest PCR amplification of the replication products was performed and the amplified products was conducted by high-throughput sequencing at last.The sequencing results showed that ALKBH2 played an important role on replication of 1m A,while ALKBH3had no significant effect on the replication process of 1m A.In this study,we mainly studied the biological effects of ALKBH2 and ALKBH3on replication of 1m A.The results showed that ALKBH2 affected the replication efficiency and played an vital role in the normal replication process of 1m A.While ALKBH3 had no significant effect on the replication efficiency and fidelity of 1m A,which may be related to the different repair mechanisms of ALKBH2 and ALKBH3 to1 mA.