Study On The Molecular Mechanism Of RCC1 And PALB2 In Regulating DNA Damage And Repair Through Histone Acetylation Repair

DNA is the most important genetic material of organisms.The integrity and stable molecular structure of DNA plays an important role in cell survival and normal physiological functions.Under the action of various factors inside and outside the organism,DNA will continue to cause various damages,but there are also perfect DNA repair mechanisms in cells to cope with these damages,thereby maintaining genetic stability.The DNA damage response is the basis for cells to maintain the stability of the genome.The defect of the DNA damage response can lead to the occurrence and development of many diseases,including tumors.The chromosome condensation regulator 1(RCC1)is the known guanine nucleotide exchange factor of Ran,which binds to the Ran protein to convert Ran-GDP into RanGTP.RCC1 plays an important role in nuclear and cytoplasmic transport,cell cycle regulation and microtubule assembly through the Ran-GTP concentration gradient.The study found that BRCA2 localization and partner and localizer of BRCA2(PALB2)interacted with breast cancer susceptibility gene 1(BRCA1)and breast cancer susceptibility gene 2(BRCA2).The complex cooperates in homologous recombination(Homologous recombination,HR)repair.PALB2 is the core protein linking BRCA1 and BRCA2,and BRCA1 is the upstream regulator in this reaction pathway.Studies have shown that PALB2 plays a very important role in the process of DNA damage that depends on BRCA1/BRCA2 and inhibits tumorigenesis.Histone acetylation can play an important role in many physiological functions of cells.In the process of DNA damage repair,the histone acetylation status has changed.Both histone acetylation and histone deacetylation are involved in the DNA double-strand break(DSB)repair process.In this study,we determined the interaction between RCC1 and PALB2 in vivo by immunoprecipitation.In order to further determine whether RCC1 is involved in DNA damage repair,laser scribing was used to induce DNA damage,and immunofluorescence staining was used to verify that RCC1 can participate in the process of DNA damage repair.After transfecting U2 OS cells with si RNA,after knocking down RCC1 and PALB2,MTT detected hypersensitivity in the case of Cisplatin(DDP)and Methyl methanesulfonate(MMS)damage.Reflected,the results showed that the cell survival rate was significantly reduced after knockdown of RCC1 and PALB2.In order to explore the mechanism by which RCC1 and PALB2 are involved in DNA damage repair pathways,we used the DR-GFP detection system.Through flow cytometry analysis,we found that RCC1 and PALB2 are involved in the homologous recombination repair pathway of DNA damage repair.After laser scribing the U2 OS cells transfected by si RNA and knockdown,immunostaining was performed.Through the staining and screening of different homologous recombination repair related proteins,it was found that RCC1 can affect the recruitment of homologous recombination repair proteins Rad51 and BRCA2.We also studied the effects of PALB2,RCC1 and PALB2 and RCC1 knockdown on histone acetylation.After knocking down PALB2,RCC1 in the cell and simultaneously knocking down the expression of PALB2 and RCC1 by si RNA,a series of changes in the expression level of acetylation of lysine at different positions on H2 A and H2 B were detected,and H2BK12 Ac,H2BK15Ac,H2BK20 Ac The expression levels of multiple acetylation sites are significantly reduced.In addition,the decreased expression of PALB2 and RCC1 can affect the killing of cells by suberoylanilide hydroxamic acid(SAHA),a deacetylase inhibitor.This project found that RCC1 is directly involved in DNA damage repair,RCC1 and PALB2 will affect the drug sensitivity of cells,and RCC1 and PALB2 jointly regulate the homologous recombination repair pathway.In addition,this study also explored the effects of RCC1 and PALB2 on DNA damage repair through histone acetylation.