Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis最新文献

Understanding the origins of mutations in tumor suppressor genes and oncogenes associated with cancers in different tissues is critical to the development of potential prevention strategies. Analysis of >10,000 nonsense mutations in 63 tumor suppressor genes based on the ratio of the number of nonsense mutations per codon type is reported for each gene. The ratio for C•G→T•A nonsense mutations at Arg CGA codons to the number of CGA codons in all cancers is 23 (3088 total nonsense mutations for 134 CGA codons in the 63 suppressor genes). The ratio for this codon, which is attributed to hydrolytic deamination of 5-methylcytosine at CpG sites based on the sequence context, is 6-fold higher than the next highest ratio that involves a C•G→T•A transition at Trp TGG codons. C•G→A•T transversions at Glu, Ser, Tyr, Gly and Cys codons account for 25 % of the total nonsense mutations but the mutation per codon ratio for these codons is 1.0. Analysis of the bases 5′ of the mutated CGA codons in the 63 tumor suppressor genes in all cancers shows a preference of 5′-G > C ∼ T ∼ A, which is not indicative of a role for enzymatic deamination by deaminases. Overall C•G→T•A mutations account for 61 % of all of the nonsense mutations in the collection of tumor suppressor genes. It is demonstrated that the ratio of C•G→T•A deamination-associated nonsense mutations at CGA codons (hydrolytic deamination) to the number of frame shift insertion/deletion mutations (i.e., replication based) for 5 major tumor suppressors genes are very similar in 3 different tissues that undergo a wide range of stem cell divisions. Therefore, the frequency of deamination mutations parallels the number of stem cell replications. This may reflect the generation of more solvent accessible single-stranded DNA regions during polymerization that are kinetically more prone to deamination.

Although UV-induced mutagenesis has been studied extensively, the precise mechanisms that convert UV-induced DNA damage into mutations remain elusive. One well-studied mechanism involves DNA polymerase (Pol) η and ζ, which produces C > T transitions during translesion synthesis (TLS) across pyrimidine dimers. We previously proposed another biochemical mechanism that involves multiple UV-irradiations with incubation in the dark in between. The incubation facilitates spontaneous deamination of cytosine in a pyrimidine dimer, and the subsequent UV irradiation induces photolyase-independent (direct) photoreversal that converts cytosine into monomeric uracil residue. In this paper, we first demonstrate that natural sunlight can induce both mutational processes in vitro. The direct photoreversal was also reproduced by monochromatic UVB at 300 nm. We also demonstrate that post-irradiation incubation in the dark is required for both mutational processes, suggesting that cytosine deamination is required for both the Pol η/ζ-dependent and the photoreversal-dependent mechanisms. Another Y-family polymerase Pol ι also mediated a mutagenic TLS on UV-damaged templates when combined with Pol ζ. The Pol ι-dependent mutations were largely independent of post-irradiation incubation, indicating that cytosine deamination was not essential for this mutational process. Sunlight-exposure also induced C > A transversions which were likely caused by oxidation of guanine residues. Finally, we constructed in vitro mutation spectra in a comparable format to cancer mutation signatures. While both Pol η-dependent and photoreversal-dependent spectra showed high similarities to a cancer signature (SBS7a), Pol ι-dependent mutation spectrum has distinct T > A/C substitutions, which are found in another cancer signature (SBS7d). The Pol ι-dependent T > A/C substitutions were resistant to T4 pyrimidine dimer glycosylase-treatment, suggesting that this mutational process is independent of cis-syn pyrimidine dimers. An updated model about multiple mechanisms of UV-induced mutagenesis is discussed.

Purpose

Radiotherapy plays a pivotal role in the treatment of cancer. One of the main challenges in this treatment modality is radiation-induced complications in some patients affected by high radiosensitivity (RS). The differences in RS are determined mainly by genetic factors. Therefore, identifying the genes and mechanisms that affect RS in different cells is essential for evaluating radiotherapy outcomes. In the present study, the ability to repair DNA double-stranded breaks (DSB) is evaluated, followed by examining the expression levels of CDKN1A (p21), cyclinD1, and Mre11 genes in human fibroblasts with different RSs.

Materials & methods

Cellular RS was measured by survival fraction at 2 Gy (SF2). The γ-H2AX assay was used for assessing DNA repair capacity. Eventually, gene expression levels from each cell line 4 and 24 h after irradiation (at 2, 4, and 8 Gy) were measured by real-time PCR.

Results

The SF2 values for the cell lines ranged from 0.286 to 0.641, and RS differences of fibroblast cells were identified. Among the studied genes, the expression of Mre11 was the most important. Analysis of the real-time PCR data showed that changes in Mre11 gene expression (4 h after 8 Gy irradiation) were directly correlated with the RS (R² = 0.905). The difference in the expression of the p21 gene (4 h after 4 Gy irradiation) was also promising. Finally, the flow cytometry analysis showed that the radioresistant cell lines quickly repaired DBS damages. However, the repair process was slow in the radiosensitive cell line, and the residual damage is significantly higher than other cell lines (P < 0.01).

Conclusions

This study indicates that changes in the expression of p21 and Mre11 genes play an important role in cell response to radiation and thus these genes can be introduced as biomarkers to predict RS in normal cell lines.

Covalent linkage between DNA and proteins produces highly toxic lesions and can be caused by commonly used chemotherapeutic agents, by internal and external chemicals and by radiation. In this study, using Escherichia coli, we investigate the consequences of 5-azacytidine (5-azaC), which traps covalent complexes between itself and the Dcm cytosine methyltransferase protein. DNA protein crosslink-dependent effects can be ascertained by effects that arise in wild-type but not in dcmΔ strains. We find that 5-azaC induces the bacterial DNA damage response and stimulates homologous recombination, a component of which is Dcm-dependent. Template-switching at an imperfect inverted repeat (“quasipalindrome”, QP) is strongly enhanced by 5-azaC and this enhancement was entirely Dcm-dependent and independent of double-strand break repair. The SOS response helps ameliorate the mutagenic effect of 5-azaC but this is not a result of SOS-induced DNA polymerases since their induction, especially PolIV, seems to stimulate QP-associated mutagenesis. Cell division regulator SulA was also required for recovery of QP mutants induced by 5-azaC. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is strongly elevated, suggesting it may play a role in DPC tolerance. Deletions at short tandem repeats, which occur likewise by a replication template-switch, are elevated, but only modestly, by 5-azaC. We see evidence for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. Whether this occurs by a different protein/DNA crosslink or by an alternative form of DNA damage is unknown

CITED2 is a transcription co-activator that interacts with TFAP2 and CBP/ P300 transcription factors to regulate the proliferation and differentiation of the cardiac progenitor cells. It acts upstream to NODAL-PITX2 pathways and regulates the left-right asymmetry. Both human genetic and model organism studies have shown that altered expression of CITED2 causes various forms of congenital heart disease. Therefore, we sought to screen the coding region of CITED2 to identify rare genetic variants and assess their impact on the structure and function of the protein. Here, we have screened 271 non-syndromic, sporadic CHD cases by Sanger’s sequencing method and detected a non-synonymous variant (c.301C>T, p.P101S) and two synonymous variants (c.21C>A, p.A7A; c.627C>G, p.P209P). The non-synonymous variant c.301C>T (rs201639244) is a rare variant with a minor allele frequency of 0.00011 in the gnomAD browser and 0.0018 in the present study. in vitro analysis has demonstrated that p.P101S mutation upregulates the expression of downstream target genes Gata4, Mef2c, Nfatc1&2, Nodal, Pitx2, and Tbx5 in P19 cells. Luciferase reporter assay also demonstrates enhanced activation of downstream target promoters. Further, in silico analyses implicate that increased activity of mutant CITED2 is possibly due to phosphorylation of Serine residue by proline-directed kinases. Homology modeling and alignment analysis have also depicted differences in hydrogen bonding and tertiary structures of wild-type versus mutant protein. The impact of synonymous variations on the mRNA structure of CITED2has been analyzed by Mfold and relative codon bias calculations. Mfold results have revealed that both the synonymous variants can alter the mRNA structure and stability. Relative codon usage analysis has suggested that the rate of translation is attenuated due to these variations. Altogether, our results from genetic screening as well as in vitro and in silico studies support a possible role of nonsynonymous and synonymous mutations in CITED2contributing to pathogenesis of CHD.

Polynucleotide kinase phosphatase (PNKP) has dual enzymatic activities as kinase and phosphatase for DNA ends, which are the prerequisite for the ligation, and thus is involved in base excision repair, single-strand break repair and non-homologous end joining for double-strand break (DSB) repair. In this study, we examined mechanisms for the recruitment of PNKP to DNA damage sites by laser micro-irradiation and live-cell imaging analysis using confocal microscope. We show that the forkhead-associated (FHA) domain of PNKP is essential for the recruitment of PNKP to DNA damage sites. Arg35 and Arg48 within the FHA domain are required for interactions with XRCC1 and XRCC4. PNKP R35A/R48A mutant failed to accumulate on the laser track and siRNA-mediated depletion of XRCC1 and/or XRCC4 reduced PNKP accumulation on the laser track, indicating that PNKP is recruited to DNA damage sites via the interactions between its FHA domain and XRCC1 or XRCC4. Furthermore, cells expressing PNKP R35A/R48A mutant exhibited increased sensitivity toward ionizing radiation in association with delayed SSB and DSB repair and genome instability, represented by micronuclei and chromosome bridges. Taken together, these findings revealed the importance of PNKP recruitment to DNA damage sites via its FHA domain for DNA repair and maintenance of genome stability.

DNA damage has been hypothesized to be a driving force of the aging process. At the same time, there exists multiple compounds that can extend lifespan in model organisms, such as yeast, worms, flies, and mice. One possible mechanism of action for these compounds is a protective effect against DNA damage. We investigated whether five of these lifespan-extending compounds, dinitrophenol, metformin, rapamycin, resveratrol, and spermidine, could protect nuclear DNA in the yeast Saccharomyces cerevisiae at the same doses under which they confer lifespan extension. We found that rapamycin and spermidine were able to decrease the spontaneous mutation rate at the CAN1 locus, whereas dinitrophenol, metformin, and resveratrol were able to protect yeast against CAN1 mutations induced by ethyl methanesulfonate (EMS). We also tested whether these compounds could enhance survival against EMS, ultraviolet (UV) light, or hydrogen peroxide (H₂O₂) insult. All five compounds conferred a protective effect against EMS, while metformin and spermidine protected yeast against UV light. Somewhat surprisingly, none of the compounds were able to afford a significant protection against H₂O₂, with spermidine dramatically sensitizing cells. We also examined the ability of these compounds to increase lifespan when growth-arrested by hydroxyurea; only spermidine was found to have a positive effect. Overall, our results suggest that lifespan-extending compounds may act in part by protecting nuclear DNA.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

Lung cancer is a prominent type of cancer, which leads to high mortality rate worldwide. The major lung cancers lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) occur mainly due to somatic driver mutations in proteins and screening of such mutations is often cost and time intensive. Hence, in the present study, we systematically analyzed the preferred residues, residues pairs and motifs of 4172 disease prone sites in 195 proteins and compared with 4137 neutral sites. We observed that the motifs LG, QF and TST are preferred in disease prone sites whereas GK, KA and ISL are predominant in neutral sites. In addition, Gly, Asp, Glu, Gln and Trp are preferred in disease prone sites whereas, Ile, Val, Lys, Asn and Phe are preferred in neutral sites. Further, utilizing deep neural networks, we have developed a method for predicting disease prone sites with amino acid sequence based features such as physicochemical properties, conservation scores, secondary structure and di and tri-peptide motifs. The model is able to predict the disease prone sites at an accuracy of 81 % with sensitivity, specificity and AUC of 82 %, 78 % and 0.91, respectively, on 10-fold cross-validation. When the model was tested with a set of 417 disease-causing and 413 neutral sites, we obtained an accuracy and AUC of 80 % and 0.89, respectively. We suggest that our method can serve as an effective method to identify the disease causing and neutral sites in lung cancer. We have developed a web server CanProSite for identifying the disease prone sites and it is freely available at-https://web.iitm.ac.in/bioinfo2/CanProSite/.