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Herein we present an assay allowing concurrent detection of oxidative DNA damage and photoproducts. We apply DNA repair enzymes specific for each lesion type to incorporate spectrally distinct fluorescent nucleotides, enabling simultaneous quantification of the lesions on individual DNA molecules. We follow the repair of both damage types in skin cells exposed to artificial sunlight.

Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end-to-end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end-to-end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end-to-end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend., (Copyright © 2018 John Wiley & Sons, Ltd.)

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5'-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1 -/- cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5'-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1 -/- and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts., (Copyright © 2018. Published by Elsevier B.V.)

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD(+) and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted., (Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone-positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancers (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression in the TCGA database and its expression and localization in TNBC cell lines. The TCGA database revealed high XRCC1 expression in TNBC tumors and TNBC cell lines show variable, but mostly high expression of XRCC1. XRCC1 localized outside of the nucleus in some TNBC cell lines, altering their ability to repair base lesions and single-strand breaks. Subcellular localization of POL β also varied and did not correlate with XRCC1 localization. Basal levels of DNA damage correlated with observed changes in XRCC1 expression, localization, and measure repair capacity. The results confirmed that XRCC1 expression changes indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with protein localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and tumors, XRCC1 expression levels should be assessed when evaluating treatment responses of TNBC preclinical model cells. [ABSTRACT FROM AUTHOR]

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer is the most diagnosed cancer among men in the United States. African American men are diagnosed with and succumb to prostate cancer at higher rates than other demographic groups. Previously published works described the biological differences in prostate tumors that may contribute to poorer outcomes in African American men compared to European American men. This study was designed to explore the DNA lesion profiles found in prostate tissues. Using tissue microarrays, we found that prostate tumors from African American patients have more uracil and pyrimidine damage, elevated UNG levels, and reduced XRCC1 levels than European American tumors, which may indicate defects in the base excision repair pathway. In addition, these men had higher UMP and lower expression of folate cycle metabolites, suggesting that metabolic rewiring may also contribute to the dysregulation of base excision repair. African Americans (AA) are two times more likely to be diagnosed with and succumb to prostate cancer (PCa) compared to European Americans (EA). There is mounting evidence that biological differences in these tumors contribute to disparities in patient outcomes. Our goal was to examine the differences in DNA damage in AA and EA prostate tissues. Tissue microarrays with matched tumor-benign adjacent pairs from 77 AA and EA PCa patients were analyzed for abasic sites, oxidative lesions, crosslinks, and uracil content using the Repair Assisted Damage Detection (RADD) assay. Our analysis revealed that AA PCa, overall, have more DNA damage than EA PCa. Increased uracil and pyrimidine lesions occurred in AA tumors, while EA tumors had more oxidative lesions. AA PCa have higher levels of UMP and folate cycle metabolites than their EA counterparts. AA PCa showed higher levels of UNG, the uracil-specific glycosylase, than EA, despite uracil lesions being retained within the genome. AA patients also had lower levels of the base excision repair protein XRCC1. These results indicate dysfunction in the base excision repair pathway in AA tumors. Further, these findings reveal how metabolic rewiring in AA PCa drives biological disparities and identifies a targetable axis for cancer therapeutics. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is used heavily in the production of polycarbonate plastics, thermal receipt paper, and epoxies. Ubiquitous exposure to BPA has been linked to obesity, diabetes, and breast and reproductive system cancers. Resistance to chemotherapeutic agents has also been shown in cancer cell models. Here, we investigated BPA's ability to confer resistance to camptothecin (CPT) in mouse embryonic fibroblasts (MEFs). MEFs are sensitive to CPT; however, co-exposure of BPA with CPT improved cell survival. Co-exposure significantly reduced Top1-DNA adducts, decreasing chromosomal aberrations and DNA strand break formation. This decrease occurs despite BPA treatment increasing the protein levels of Top1. By examining chromatin structure after BPA exposure, we determined that widespread compaction and loss of nuclear volume occurs. Therefore, BPA reduced CPT activity by reducing the accessibility of DNA to Top1, inhibiting DNA adduct formation, the generation of toxic DNA strand breaks, and improving cell survival. [ABSTRACT FROM AUTHOR]