damaged dna
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Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

Abstract Background Doxorubicin induces DNA damage not only in tumor cells but also in the cardiomyocyte, and accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. We previously found that cardiomyocyte-specific deletion of SIRT1, a NAD+-dependent histone/protein deacetylase, worsens doxorubicin-induced cardiotoxicity in mice. However, its molecular mechanism remains unclear. Phosphorylation of histone H2AX at Ser139 catalyzed by ATM (mutated in ataxia-telangiectasia) at the sites of DNA damage is a critical mediator for DNA repair. Purpose Here, we tested the hypothesis that deacetylation of H2AX by SIRT1 mediates DNA damage response to counteract doxorubicin-induced cardiotoxicity. Methods and results Wild-type (WT) mice and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1-cKO) mice at 3 month of age received doxorubicin (4 IP injections of 5 mg/kg/week) or a vehicle. Immunoblotting of myocardial lysates from mice 1 week after final doxorubicin showed that doxorubicin increased phospho-Ser139-H2AX level by 1.6-fold in WT, but such a response was blunted in SIRT1-cKO. Ser1981-phosphorylations of ATM induced by doxorubicin were similar in WT and SIRT1-cKO. DNA fragmentation evaluated by TUNEL staining revealed that the increase in TUNEL-positive nuclei by doxorubicin was more in SIRT1-cKO (0.13% to 0.38%) than those in WT (0.07% to 0.19%), suggesting higher DNA damage in SIRT1-cKO. In H9c2 cardiomyocytes, knockdown of SIRT1 also abolished the doxorubicin-induced Ser139-phosphorylation of H2AX without changing phospho-ATM levels. Increases in DNA damage evaluated by comet assay and cleavage of caspase-3 by doxorubicin were also enhanced in SIRT1-knockdown cells. Immunostaining for acetyl-Lys5-H2AX in the heart sections revealed that acetyl-Lys5-H2AX levels were increased in SIRT1-cKO by 58% compared with those in WT. In H9c2 cells, acetyl-Lys5-H2AX level was also increased by SIRT1 knockdown and reduced by expression of wild-type SIRT1. To test the role of the increased acetyl-Lys5-H2AX level under SIRT1 inhibition, we generated a mutant in which Lys5 was substituted to glutamine (K5Q H2AX) as a mimic of acetylated Lys5. In COS7 cells expressing WT or K5Q H2AX, Ser139-phosphorylation induced by doxorubicin was suppressed in K5Q mutant. In addition, doxorubicin-induced cleavage of caspase-3 was enhanced in H9c2 cells expressing K5Q H2AX as well as S139A H2AX, that cannot be phosphorylated at Ser139, compared with cells expressing WT H2AX. Conclusions These findings suggest that the increased Lys5 acetylation of H2AX via SIRT1 inhibition interferes Ser139 phosphorylation, leading to accumulation of damaged DNA and promotion of the apoptotic response. Such regulation of the DNA damage response contributes to protection by SIRT1 against doxorubicin-induced cardiotoxicity. FUNDunding Acknowledgement Type of funding sources: None.

Uracil-DNA glycosylase (UDG) is one of the most important base excision repair (BER) enzymes involved in the repair of uracil-induced DNA lesion by removing uracil from the damaged DNA. Uracil in DNA may occur due to cytosine deamination or deoxy uridine monophosphate (dUMP) residue misincorporation during DNA synthesis. Medical evidences show that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, and liver cancer. Therefore, the research of UDG is crucial in cancer treatment and prevention as well as other clinical activities. Here we applied multiple computational methods to study UDG in several perspectives: Understanding the stability of the UDG enzyme in different pH conditions; studying the differences in charge distribution between the pocket side and non-pocket side of UDG; analyzing the field line distribution at the interfacial area between UDG and DNA; and performing electrostatic binding force analyses of the special region of UDG (pocket area) and the target DNA base (uracil) as well as investigating the charged residues on the UDG binding pocket and binding interface. Our results show that the whole UDG binding interface, and not the UDG binding pocket area alone, provides the binding attractive force to the damaged DNA at the uracil base.

Abstract Background:Head and neck cancers account for about 30% of all cancers in India. The incidence rates of HNSCC in India are30/1, 00,000 for males and 10/1, 00,000 for females. The commonly used treatment modalities include surgery, chemotherapy and radiotherapy. Studies conducted in different types of cancers showed that there is anincreased primary DNA damage even before the commencement of treatment in cancer patients.The treatment modality will further induce DNA damage in addition to the already existing DNA damage.In normal healthy people, DNA damage is effectively repaired. However, in patients with carcinoma, chemo-radiation induced DNA damage is not repaired so effectively. Consequently, there is a high risk of secondary carcinoma by unrepaired damaged DNA.Methodology:In this study, the degree of DNA damage is assessed by comet assay technique in patients with head and neck carcinoma receiving radiotherapy and had complete regression of tumor following radiotherapy. The degree of DNA damage is compared according to the age, gender and associated risk factors of the patients.Results:The comet length parameter of post-RT sample is increased when compared to baseline sample. The head diameter parameter of post-RT sample is increased when compared to baseline sample. The percentage of DNA in head parameter of post-RT sample is decreased when compared to baseline sample. The tail length parameter of post-RT sample is increased when compared to baseline sample. All these findings are indicative of DNA damage following radiotherapy. Consequently, there is a high risk of secondary carcinoma by unrepaired damaged DNA.Conclusion:Patients with locally advanced head and neck carcinoma with complete tumor response following radiotherapy showed a sequential increase in the DNA damage. The co-existing risk factors and old age may increase the baseline DNA damage in the patients with head and neck cancers.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

AbstractWhen detecting DNA profiles from forensic materials, it is pivotal to know the extent of degradation and which DNA marker can be genotyped. Ultraviolet (UV) is one of the common external factors that causes DNA damage, through which, an attempt to reveal cardinal genetic information can be made. In this study, after irradiation with three different UV wavelengths, UV-damaged DNA in the bloodstains was analyzed with long and short TaqMan assays using real-time PCR. In addition, both short tandem repeat (STR) profiles and single nucleotide polymorphisms (SNPs) from the damaged DNA at different stages of UV exposure were also assessed. With increasing in UV irradiation cycles, there was a delay of the amplification curves accompanied with a decrease in the DNA amounts collected. Despite the amplification of STR genotype was not altered after 75 cycles of UVC irradiation, all 12 SNP loci could still be detected. Furthermore, a short-assay line was detected in the absence of an amplification of the evaluation curve. The results indicate that, although the DNA template might not be useful and suitable for analysis of STR profile, this approach is of some values in detecting SNPs.