Establishment Of DSB Model In Mammalian Cells And Its Recruitment Of Histone H2A.Z

DNA double-strand break(DSB)is one of the most serious forms of injury,and many exogenous and endogenous factors can cause DSB damage in DNA.After long-term evolution,eukaryotic cells have formed a complex and sophisticated DNA repair system to deal with DNA damage.Failure to repair DSB can lead to serious consequences such as cell death,genetic diseases,and tumorigenesis.Eukaryotic cells mainly contain two major repair methods: homologous recombination(HR)and non-homologous end joining(NHEJ).DNA is the main genetic material of eukaryotes and exists in the form of chromatin in the nucleus.The nucleosome is the basic unit of chromatin,which is double-entangled on histone octamer by about 147 bp of DNA.Each histone octamer consists of two H2 A,H2B heterodimers and a tetramer of H3,H4.Numerous nucleosomes are joined by protein H1 and then gradually compressed into 30 nm filaments,which are further compressed to form a chromatin-like structure.H2 AX and H2 A.Z are two variants of histone H2 A that are widely distributed in eukaryotic nucleosomes.When the DNA double strand breaks in the chromosome,H2 A.X near the breakpoint is phosphorylated.This phosphorylated H2 A.X is specifically recognized by DNA repair enzymes and localizes the repair enzyme to the DNA damage site.In addition to the DNA replication process,H2 A.Z can also exert specific biological functions by entering/extracting nucleosomes in specific regions of chromatin or driven by specific signals.H2 A.Z recruitment is usually present in the promoter region,and as transcription is activated,H2 A.Z is also replaced by high frequency nucleosomes.The laboratory has preliminary data and ongoing studies on chromatin remodeling enzymes INO80 and DNA-PK kinase.These two complexes are closely related to ?-H2 AX and H2 A.Z and other repair proteins.The main task in this paper is to construct a DSB injury model by constructing laser in Hela cells and to detect?-H2 AX and H2 A.Z in the U20S-AsiSI-ER cell line with no change in repair time.In the Hela cells,the laser conditions causing DSB were explored and the changes of H2 A.Z were observed by immunofluorescence.The changes of ?-H2 AX and H2 A.Z were first observed by immunofluorescence staining in U20S-AsiSI-ER,and then passed through chip.The experiment further verified the results.For the future experiments to do the pre-conditions,leave spare.