In addition to DNA repair pathways,cells utilize translesion DNA synthesis(TLS)to bypass DNA lesions during replication.During TLS,Y-family DNA polymerase(Polη,Polκ,Polιand Rev1)inserts specific nucleotide opposite...In addition to DNA repair pathways,cells utilize translesion DNA synthesis(TLS)to bypass DNA lesions during replication.During TLS,Y-family DNA polymerase(Polη,Polκ,Polιand Rev1)inserts specific nucleotide opposite preferred DNA lesions,and then Polζ consisting of two subunits,Rev3 and Rev7,carries out primer extension.Here,we report the complex structures of Rev3-Rev7-Rev1^(CTD) and Rev3-Rev7-Rev1^(CTD)-Polκ^(RIR).These two structures demonstrate that Rev1^(CTD) contains separate binding sites for Polκand Rev7.Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1.We also verified through FRET experiment that Rev1,Rev3,Rev7 and Polκ form a stable quaternary complex in vivo,thereby suggesting an efficient switching mechanism where the“inserter”polymerase can be immediately replaced by an“extender”polymerase within the same quaternary complex.展开更多
N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replicati...N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.展开更多
Aim:The transcription factor RIP140(receptor interacting protein of 140 kDa)is involved in intestinal tumorigenesis.It plays a role in the control of microsatellite instability(MSI),through the regulation of MSH2 and ...Aim:The transcription factor RIP140(receptor interacting protein of 140 kDa)is involved in intestinal tumorigenesis.It plays a role in the control of microsatellite instability(MSI),through the regulation of MSH2 and MSH6 gene expression.The aim of this study was to explore its effect on the expression of POLK,the gene encoding the specialized translesion synthesis(TLS)DNA polymeraseκknown to perform accurate DNA synthesis at microsatellites.Methods:Different mouse models and engineered human colorectal cancer(CRC)cell lines were used to analyze by RT-qPCR,while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression.Published DNA microarray datasets were reanalyzed.The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined.Results:RIP140 positively regulates,at the transcriptional level,the expression of the POLK gene,and this effect involves,at least partly,the p53 tumor suppressor.In different cohorts of CRC biopsies(with or without MSI),a strong positive correlation was observed between RIP140 and POLK gene expression.In connection with its effect on POLK levels and the TLS function of this polymerase,the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene.Finally,the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK,thus strengthening the functional link between the two genes in human CRC.Conclusion:The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability,and more generally to the control of genome integrity.展开更多
基金supported financially by the National Basic Research Program(973 Program)(No.2011CB910302)the National Natural Science Foundation of China(Grant Nos.31025009,31021062 and 31200558).
文摘In addition to DNA repair pathways,cells utilize translesion DNA synthesis(TLS)to bypass DNA lesions during replication.During TLS,Y-family DNA polymerase(Polη,Polκ,Polιand Rev1)inserts specific nucleotide opposite preferred DNA lesions,and then Polζ consisting of two subunits,Rev3 and Rev7,carries out primer extension.Here,we report the complex structures of Rev3-Rev7-Rev1^(CTD) and Rev3-Rev7-Rev1^(CTD)-Polκ^(RIR).These two structures demonstrate that Rev1^(CTD) contains separate binding sites for Polκand Rev7.Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1.We also verified through FRET experiment that Rev1,Rev3,Rev7 and Polκ form a stable quaternary complex in vivo,thereby suggesting an efficient switching mechanism where the“inserter”polymerase can be immediately replaced by an“extender”polymerase within the same quaternary complex.
基金supported by the National Natural Science Foundation of China (Nos. 21807030, 21907028)the Science and Technology Innovation Program of Hunan Province(No. 2019RS2020)+1 种基金Natural Science Foundation of Hunan Province(No. 2020JJ5046)the Fundamental Research Funds for the Central Universities (Nos. 531118010061, 531118010259)。
文摘N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.
文摘Aim:The transcription factor RIP140(receptor interacting protein of 140 kDa)is involved in intestinal tumorigenesis.It plays a role in the control of microsatellite instability(MSI),through the regulation of MSH2 and MSH6 gene expression.The aim of this study was to explore its effect on the expression of POLK,the gene encoding the specialized translesion synthesis(TLS)DNA polymeraseκknown to perform accurate DNA synthesis at microsatellites.Methods:Different mouse models and engineered human colorectal cancer(CRC)cell lines were used to analyze by RT-qPCR,while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression.Published DNA microarray datasets were reanalyzed.The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined.Results:RIP140 positively regulates,at the transcriptional level,the expression of the POLK gene,and this effect involves,at least partly,the p53 tumor suppressor.In different cohorts of CRC biopsies(with or without MSI),a strong positive correlation was observed between RIP140 and POLK gene expression.In connection with its effect on POLK levels and the TLS function of this polymerase,the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene.Finally,the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK,thus strengthening the functional link between the two genes in human CRC.Conclusion:The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability,and more generally to the control of genome integrity.