Description: Publication date: June–July 2018 Source: DNA Repair, Volumes 66–67 Author(s): Guillermo de Cárcer, Pablo Huertas, Andres J. López-Contreras The International University of Andalusian (UNIA) held on the 13th to 15th of November 2017 was a meeting oriented to the concept of Chromosomal Instability and related diseases. The meeting was part of the renowned UNIA workshops programme “Current Trends in Biomedicine”, held in the UNESCO World Heritage awarded city Baeza, located in the south of Spain. The goal of this Workshop was to bring together experts in DNA repair and chromosome segregation in order to address the phenomenon of Chromosomal Instability as a whole, facilitating the communication between scientists from different fields to foster cross-disciplinary approaches. This report summarizes a selection of the many interesting results and data presented during the meeting.

Description: Publication date: Available online 5 May 2018 Source: DNA Repair Author(s): Marcus C. Parrish, Isaac A. Chaim, Zachary D. Nagel, Steven R. Tannenbaum, Leona D. Samson, Bevin P. Engelward Nitric oxide, a reactive nitrogen species released by lymphocytes during inflammation, has been shown to react with DNA, proteins, and cells. Protein S-nitrosation, the process by which nitric oxide reacts with cysteine residues on proteins, has been found to modulate DNA repair. However, relatively little is known about the role of S-nitrosation in the context of repair of alkylation damage by the base excision repair pathway (BER). BER of DNA exposed to a methylating agent, such as methylmethane sulfonate (MMS), is initiated by the Alkyladenine DNA Glycosylase (AAG), which removes damaged bases. Here, we analyzed the effects of the transnitrosating peptide S-nitrosoglutathione (GSNO) on the repair of methylated bases. Through the use of the CometChip, a high-throughput version of the comet assay that measures DNA strand breaks, we observed an AAG-dependent increase in BER intermediates in GSNO-exposed mouse embryonic fibroblasts after MMS challenge. Through the use of the Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR), a high-throughput assay used to measure DNA repair capacity, GSNO exposure was found to alter the activities of BER proteins and reduce overall BER capacity. Furthermore, cells exposed to both GSNO and MMS displayed reduced viability. Given that unrepaired BER intermediates are toxic, these results suggest that the altered activities of BER proteins following GSNO exposure induces an increase in BER intermediates that leads to increased cell death. Taken together, this study shows the detrimental effects of S-nitrosation on the BER pathway and reveals the deleterious impact of altered BER capacity under conditions of co-exposure to an alkylating agent.

Description: Publication date: Available online 2 May 2018 Source: DNA Repair Author(s): Evgeny Kiselev, Thomas S. Dexheimer, Christophe Marchand, Shar-yin Naomi Huang, Yves Pommier Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an ubiquitous DNA repair enzyme present in yeast, plants and animals. It removes a broad range of blocking lesions at the ends of DNA breaks. The catalytic core of TDP1 consists in a pair of conserved histidine-lysine-asparagine (HKN) motifs. Analysis of the human TDP1 (hTDP1) crystal structure reveals potential involvement of additional residues that shape the substrate binding site. In this biochemical study, we analyzed four such conserved residues, tyrosine 204 (Y204), phenylalanine 259 (F259), serine 400 (S400) and tryptophan 590 (W590). We show that the F259 residue of hTDP1 is critical for both 3′- and 5′-phosphodiesterase catalysis. We propose that the double π-π interactions of the F259 residue with the −2 and −3 nucleobases serve to position the nucleopeptide substrate in phase with the active site histidines of hTDP1. Mutating Y204 of hTDP1 to phenylalanine (Y204F), as in fly and yeast TDP1 enzymes, had minor impact on TDP1 activity. In constrast, we find that S400 enhances 3′-processing activity while it suppresses 5′-processing activity, thereby promoting specificity for 3′-substrates. W590 is selectively important for 5′-processing. These results reveal the impact of conserved amino acid residues that participate in defining the DNA binding groove around the dual HKN catalytic core motif of TDP1, and their differential roles in facilitating the 3′- vs 5′-end processing activities of hTDP1.

Description: Publication date: Available online 26 April 2018 Source: DNA Repair Author(s): Raima Das, Sharbadeb Kundu, Shaheen Laskar, Yashmin Choudhury, Sankar Kumar Ghosh Head and neck cancer (HNC), the sixth most common cancer globally, stands second in India. In Northeast (NE) India, it is the sixth most common cause of death in males and seventh in females. Prolonged tobacco and alcohol consumption constitute the major etiological factors for HNC development, which induce DNA damage. Therefore, DNA repair pathway is a crucial system in maintaining genomic integrity and preventing carcinogenesis. The present work was aimed to predict the consequence of significant germline variants of the DNA repair genes in disease predisposition. Whole exome sequencing was performed in Ion Proton™ platform on 15 case-control samples from the HNC-prevalent states of Manipur, Mizoram, and Nagaland. Variant annotation was done in Ion reporter as well as wANNOVAR. Subsequent statistical and bioinformatics analysis identified significant exonic and intronic variants associated with HNC. Amongst our observed variants, 78.6% occurred in ExAC, 94% reported in dbSNP and 5.8% & 9.3% variants were present in ClinVar and HGMD, respectively. The total variants were dispersed among 199 genes with DSBR and FA pathway being the most mutated pathways. The allelic association test suggested that the intronic variants in HLTF and RAD52 gene significantly associated (P 〈 0.05) with the risk (OR > 5), while intronic variants in PARP4, RECQL5, EXO1 and PER1 genes and exonic variant in TDP2 gene showed protection (OR 〈 1) for HNC. MDR analysis proposed the exonic variants in MSH6, BRCA2, PALB2 and TP53 genes and intronic variant in RECQL5 genetic region working together during certain phase of DNA repair mechanism for HNC causation. In addition, other intronic and 3'UTR variations caused modifications in the transcription factor binding sites and miRNA target sites associated with HNC. Large-scale validation in NE Indian population, in-depth structure prediction and subsequent simulation of our recognized polymorphisms is necessary to identify true causal variants related to HNC.

Description: Publication date: Available online 27 April 2018 Source: DNA Repair Author(s): Nathaniel W. Holton, Yuval Ebenstein, Natalie R. Gassman Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, R epair A ssisted D amage D etection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity. Graphical abstract

Description: Publication date: Available online 23 April 2018 Source: DNA Repair Author(s): Amar Desai, Yan Yan, Stanton L. Gerson

Description: Publication date: Available online 22 April 2018 Source: DNA Repair Author(s): Rosario Prados-Carvajal, Ana López-Saavedra, Cristina Cepeda-García, Sonia Jimeno, Pablo Huertas The appropriate repair of DNA double strand breaks is critical for genome maintenance. Thus, several cellular pathways collaborate to orchestrate a coordinated response. These include the repair of the breaks, which could be achieved by different mechanisms. A key protein involved in the regulation of the repair of broken chromosomes is CtIP. Here, we have found new partners of CtIP involved in the regulation of DNA break repair through affecting DNA end resection. We focus on the splicing complex SF3 B and show that its depletion impairs DNA end resection and hampers homologous recombination. Functionally, SF3 B controls CtIP function at, as least, two levels: by affecting CtIP mRNA levels and controlling CtIP recruitment to DNA breaks, in a way that requires ATM-mediated phosphorylation of SF3B2 at serine 289. Indeed, overexpression of CtIP rescues the resection defect caused by SF3 B downregulation. Strikingly, other SF3 B depletion phenotypes, such as impaired homologous recombination or cellular sensitivity to DNA damaging agents, are independent of CtIP levels, suggesting a more general role of SF3 B in controlling the response to chromosome breaks.

Description: Publication date: Available online 21 April 2018 Source: DNA Repair Author(s): Maryam Majidinia, Saber Ghazizadeh Darband, Mojtaba Kaviani, Seyed Mohammad Nabavi, Rana Jahanban-Esfahlan, Bahman Yousefi Despite their simple structure, the Notch family of receptors regulates a wide-spectrum of key cellular processes including development, tissue patterning, cell-fate determination, proliferation, differentiation and, cell death. On the other hand, accumulating date pinpointed the role of non-coding microRNAs, namely miRNAs in cancer initiation/progression via regulating the expression of multiple oncogenes and tumor suppressor genes, as such the Notch signaling. It is now documented that these two partners are in one or in the opposite directions and rule together the cancer fate. Here, we review the current knowledge relevant to this tricky interplay between different miRNAs and components of Notch signaling pathway. Further, we discuss the implication of this crosstalk in cancer progression/regression in the context of cancer stem cells, tumor angiogenesis, metastasis and emergence of multi-drug resistance. Understanding the molecular cues and mechanisms that occur at the interface of miRNA and Notch signaling would open new avenues for development of novel and effective strategies for cancer therapy.

Description: Publication date: May 2018 Source: DNA Repair, Volume 65

Description: Publication date: Available online 17 April 2018 Source: DNA Repair Author(s): Shiladitya Sengupta, Chunying Yang, Muralidhar L. Hegde, Pavana M. Hegde, Joy Mitra, Arvind Pandey, Arijit Dutta, Abdul Tayyeb Datarwala, Kishor K. Bhakat, Sankar Mitra Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1’s disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296–298 with neutral Ala lost nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites did not affect NEIL1’s nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.