Study On Idenfication And Fuction Of MARVELD1 PARylation Site

MARVELD1 is a multi-functional protein,located in human chromosome 10 long arm 2 region 4 Band 2 sub band(10q24.2).The full length of MARVELD1 is3238 bp,and its molecular weight is about 18.9 k Da.Previous data analysis suggested that MARVELD1 could maintain the stability of the genome and reduce the level of DNA damage in cells after DNA damage drugs treatment.Further studies confirmed that there was a strong interaction between MARVELD1 and PARP1.PARP1 could transfer the ADP ribose unit to MARVELD1,resulting in PARylation modification of MARVELD1.However,the specific modification sites and their effects on protein function are still unclear.Based on the above results,this study mainly discussed the PARylation site of MARVELD1 under DNA damage stress and its biological role.In this study,He La cells and HEK293 T cells were used as cell model.The recombinant plasmid containing single point mutation of PARylation site was transiently transfected into He La cells and HEK293 T cells.The cells were treated with hydroxyurea(HU)and other drugs to stimulate DNA damage.Western blot analysis showed that the main PARylation sites of MARVELD1 were D102,D118 and D130.On this basis,we further explored the effect of PARylation modification level of MARVELD1 on its protein function.Combined with immunofluorescence analysis,the results showed that MARVELD1 had bright dot like aggregation in the nucleus of cells treated with hydroxyurea.The results of cell component separation also confirmed that the content of MARVELD1 in nucleus increased after hydroxyurea treatment.These results suggest that MARVELD1 is recruited into the nucleus under DNA damage stress.When PARP1 inhibitor olaparib was used to inhibit the level of PARylation modification,or PARylation modification sites(D102,D118 and D130)of MARVELD1 were co-mutated,the responsive entry of MARVELD1 was significantly reduced.These results further suggest that PARylation modification is involved in the responsive entry of MARVELD1 to DNA damage stress.In order to clarify whether PARylation modification of MARVELD1 affects the response to DNA damage,comet electrophoresis showed that the level of DNA damage increased after PARylation modification of MARVELD1 was deleted.After He La cells were treated with three kinds of DNA damage inducing drugs,hydroxyurea,camptothecin(CPT)and alfediamycin(APH),parylation level of MARVELD1 could affect cell viability.He La cells overexpressing wild-type marveld1 were compared with He La cells lacking marveld1 modification,the former is more resistant to DNA damage drugs.When PARylation modification of MARVELD1 was deleted,the degree of chromatin looseness increased significantly,and the genomic DNA was unstable,indicating that PARylated MARVELD1 affected the degree of chromatin looseness in DNA damaged sites.In conclusion,this study confirmed that aspartic acid at 102,118 and 130 of marveld1 was the main site of parylation modification.PARylated MARVELD1 can be recruited into the nucleus under the stimulation of DNA damage,reduce the level of DNA damage,improve the stress ability of cells to DNA damage stimulation,and participate in the regulation of DNA damage response.