Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.

Ganoboninketal C from Ganoderma boninense improves the efficacy of CDDP-based chemotherapy through inhibiting translesion DNA synthesis

Translesion DNA synthesis(TLS)can bypass DNA lesions caused by chemotherapeutic drugs,which usually result in drug resistance.Given its key role in mutagenesis and cell survival after DNA damage,inhibition of the TLS pathway has emerged as a potential target for improving the efficacy of DNA-damaging agents such as cisplatin(CDDP),a widely used anticancer agent.Unfortunately,few suitable natural TLS inhibitors have been reported.Here,we found that a triterpenoid compound Ganoboninketal C(26-3)from Ganoderma boninense,a traditional Chinese medicine,can impair CDDP-induced TLS polymerase eta(Polη)focus formation,PCNA monoubiquitination as well as mutagenesis.Moreover,26-3 can significantly sensitize tumor cells to CDDP killing and reduce the proportion of cancer stem cells in AGS and promote apoptosis after CDDP exposure.Interestingly,26-3 can also sensitize tumor cells to Gefitinib therapy.Mechanistically,through RNA-seq analysis,we found that 26-3 could abrogate the CDDP-induced upregulation of Polηand PIDD(p53-induced protein with a death domain),2 known factors promoting TLS pathway.Furthermore,we found that activating transcription factor 3 is a potential novel TLS modulator.Taken together,we have identified a natural TLS inhibitor 26-3,which can be potentially used as an adjuvant to improve clinical efficacy.

SEARCH FOR TRANSLESION SYNTHESIS MODULATORS FROM NATURAL PRODUCTS:ANALYTICAL DEVELOPMENT, VALIDATION AND APPLICATIONS

DNA translesion synthesis(TLS)is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis.TLS DNA polymerases pursue replication by inserting a base opposite this lesion,correctly or incorrectly

Expression of Polt in tissues and cell lines of transitional cell carcino-ma

Objective：To explore the expression of DNA polymerase iota in transitional cell carcinoma ceils and tissues; Methods： RT-PCR was applied to detect the expression of polymerase iota in BIU87 and T24 ceils, then the expression of polymerase iota was also detected in the same way in transitional cell car- cinoma which was derived from clinical bladder carcinoma and renal pelvic carcinoma. Results ： The expres- sion of Poh was low in bladder normal membrana mucosa but significantly elevated in transitional cell car- cinoma ceils. Compared with the expression of polymerase iota in bladder normal mucous membranes, the expression of polymerase iota was significantly increased in transitional cell carcinoma tissue （P〈0. 01 ） and associated with the grade of transitional cell carcinoma. Conclusion： The significantly increased ex- pression of polymerase iota may be associated with the generation and development of transitional cell carcinoma, even with its high heterogenicity.

Molecular mechanisms of DNA lesion and repair during antibody somatic hypermutation

Antibody diversification is essential for an effective immune response,with somatic hypermutation(SHM)serving as a key molecular process in this adaptation.Activation-induced cytidine deaminase(AID)initiates SHM by inducing DNA lesions,which are ultimately resolved into point mutations,as well as small insertions and deletions(indels).These mutational outcomes contribute to antibody affinity maturation.The mechanisms responsible for generating point mutations and indels involve the base excision repair(BER)and mismatch repair(MMR)pathways,which are well coordinated to maintain genomic integrity while allowing for beneficial mutations to occur.In this regard,translesion synthesis(TLS)polymerases contribute to the diversity of mutational outcomes in antibody genes by enabling the bypass of DNA lesions.This review summarizes our current understanding of the distinct molecular mechanisms that generate point mutations and indels during SHM.Understanding these mechanisms is critical for elucidating the development of broadly neutralizing antibodies(bnAbs)and autoantibodies,and has implications for vaccine design and therapeutics.

Structural insights into the assembly of human translesion polymerase complexes

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.

Next-generation sequencing-based analysis of the effect of N^(6)-methyldeoxyadenosine modification on DNA replication in human cells

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.

RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells

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.