Molecular Mechanisim Of The Expression Of DNA Replication Key Protein MCM Regulated By P53^N236S

Werner syndrome（WS）is a rare autosomal recessive disorder caused by mutations in the Wrn gene.Dr.Sandy Chang successfully established the mouse model of WS for the first time by double knockout of the mouse telomerase template Terc and Wrn genes（m TR-/-Wrn-/-,DM）.In an early study,the laboratory found that tumor cells derived from WS-background mice have an identical p53 mutation point,p53^N236S（p53S）,which is caused by mutation of asparagine at position 236 of the p53 DNA-binding region to serine.This mutation in p53 is based on the absence of telomerase,and the main function of telomerase is to extend telomeres,suggesting that p53 S is mediated by telomere damage.In order to study the functional acquisition of p53 S,a pre-laboratory study was performed to hybridize p53 S with WS mice to obtain a triple genotype heterozygous mouse,and the first occurrence of the m TR-/-gene in a triple genotype heterozygous mouse was called For G1 generation G1 m TR-/-Wrn-/-p53SS（G1TM）mice,other generations of mice were obtained by sequential hybridization.TM mice have one more p53 S background than DM mice.By comparing the same algebraic TM and DM mice,and by Ch IP-on-chip and RNA-Seq data analysis,MCM（Mini-chromosome maintenance）pathway was obtained.Related genes.In the feeding process of WS mice（DM mice）and triple genotype mice（TM mice）,the lifespan of DM mice of G4 and G5 generations was significantly shortened,while the lifespan of G4 and G5 generation mice was relatively small compared with DM.The mice have prolonged,and at the same time,there are different frequency of tumorigenesis in the G1-G3 generation TM mice.Because the telomerase activity in this tumor cell is negative,it belongs to ALT（Alternative lenthing of telomere）tumor.We verified the MCM pathway-related genes by real-time PCR.It was found that the m RNA expression of MCM pathway-related genes was higher in DM cells than in DM cells in the same generation of mice compared with G3 TM,G5DM and G5 TM,including MCM gene.The MCM protein transcriptionally translated MCM protein is one of the DNA replication permitting factors,and the MCM2-MCM7 protein usually III forms an isohexameric cyclic complex whose main function is the melting of DNA replication initiation and DNA extension.This subject mainly explores the molecular mechanism of p53 S regulating the expression of key protein MCM in DNA replication.First,real-time PCR and Western blot experiments were performed to detect m RNA and protein levels of MCM genes in different algebraic mouse cells containing p53S（TM）and no p53S（DM）.MEFs were found in the same algebraic mice.In the cells,the m RNA expression and protein expression of TM cells were higher than those of DM cells.Subsequently,in view of the complexity of the genetic background of DM and TM mice,we verified the expression of MCM gene in p53 SS,p53-/-MEF cells and p53 SS,p53-/-tumor cells,and found that MCM contained p53 S.The expression of m RNA and protein in cells were higher than those without p53 S,which indicated that p53 S has a certain regulatory effect on MCM gene.It was confirmed by Ch IP experiment that p53 S can bind to the promoter region of MCM2 gene.Combined with real-time PCR and Western blot experiments and Ch IP results,we concluded that p53 S can regulate MCM2 gene and promote MCM expression.Subsequently,we used FISH experiments to examine the telomere content of these MCM-expressing cells.It was found that in the G3 generation of DM and TM cells,there were telomere fluorescence signals;since G5 DM mice had premature aging Types of mice,normal G5 DM cells,show senescence very quickly during passage,so we barely see fluorescent signals in normal G5 DM cells,and G5 TM cells can clearly see fluorescent signals,suggesting that p53 S may be The length of the telomeres was maintained by MCM.Finally,we verified the regulation of p53 S in telomerase-deficient tumor cell-ALT tumor cells;Ch IP assay showed that p53 S is still able to bind to the promoter region of MCM2 gene in tumor cells,but in Western blot The results of the experiment showed that MCM protein was highly expressed only in G3 TM tumor cells,indicating that p53 S has a significant regulatory effect in tumor cells of G3 mice.It is worth noting that there is no significant change in the expression of MCM in G1 TM and G2 TM tumor cells.This may be due to the fact that the telomere length of G1 TM and G2 TM mice is long enough and the telomere maintenance mechanism has not been initiated,which suggests that The molecular mechanism by which p53 S regulates MCM may be affected by telomere length.We also looked at the chromosomal status of ALT tumor cells by karyotyping.We found that in ALT tumor cells,a higher proportion of chromosome breaks and fusions were found,and double microsomes were found,indicating that ALT tumors The chromosome is quite turbulent.In view of the complexity of p53 S function,we compared the occurrence of Anaphase brige in DM,TM MEF cells and TM tumor cells of the same algebraic mouse,and found that the proportion of late bridges in TM cells containing p53 S DM cells significantly higher than the same algebraic mice,suggesting that p53 S causes chromosome aberrations.In summary,first,in the MCM pathway,p53 S binds to the promoter region of the MCM2 gene to promote MCM expression;second,p53 S maintains the length of telomeres,which may be dependent on the regulation of MCM;Third,p53 S causes chromosome disorders.