The roles of fission yeast exonuclease 5 in nuclear and mitochondrial genome stability Publication date: November 2019 Source: DNA Repair, Volume 83 Author(s): Justin L. Sparks, Kimberly J. Gerik, Carrie M. Stith, Bonita L. Yoder, Peter M. Burgers Abstract The Exo5 family consists of bi-directional, single-stranded DNA-specific exonucleases that contain an iron-sulfur cluster as a structural motif and have multiple roles in DNA metabolism. S. cerevisiae Exo5 is essential for mitochondrial genome maintenance, while the human ortholog is important for nuclear genome stability and DNA repair. Here, we identify the Exo5 ortholog in Schizosaccharomyes pombe (spExo5). The activity of spExo5 is highly similar to that of the human enzyme. When the single-stranded DNA is coated with single-stranded DNA binding protein RPA, spExo5 become a 5′-specific exonuclease. Exo5Δ mutants are sensitive to various DNA damaging agents, particularly interstrand crosslinking agents. An epistasis analysis places exo5+ in the Fanconi pathway for interstrand crosslink repair. Exo5+ is in a redundant pathway with rad2+, which encodes the flap endonuclease FEN1, for mitochondrial genome maintenance. Deletion of both genes lead to severe depletion of the mitochondrial genome, and defects in respiration, indicating that either spExo5 or spFEN1 is necessary for mitochondrial DNA metabolism.

Multisystem analyses of two Cockayne syndrome associated proteins CSA and CSB reveal shared and unique functions Publication date: November 2019 Source: DNA Repair, Volume 83 Author(s): Zhenzhen Wu, Xun Zhu, Qian Yu, Yingying Xu, Yuming Wang Abstract Mutations in the CSA and CSB genes are causative of Cockayne syndrome neurological disorder. Since the identification of indispensable functions of these two proteins in transcription-coupled repair and restoring RNA synthesis following DNA damage, the paradoxical less severe clinical symptoms reported in some CS-A patients have been puzzling. In this study we compared the effects of a CSA or a CSB defect at the levels of the cell and the intact organism. We showed that CSA-deficient zebrafish embryos exhibited modest hypersensitive to UV damage than CSB depletion. We found that loss of CSA can effectively release aggregation of mutant crystallin proteins in vitro. We described the opposite effect of CSA and CSB on neuritogenesis and elucidated the differentiated gene expression pathways regulated by these two proteins. Our data demonstrate convergent and divergent roles for CSA and CSB in DNA repair and transcription regulation and provide potential explanations for the observed differences between CS-A and CS-B patients.

Interactions of high mobility group box protein 1 (HMGB1) with nucleic acids: Implications in DNA repair and immune responses Publication date: November 2019 Source: DNA Repair, Volume 83 Author(s): Pooja Mandke, Karen M. Vasquez Abstract High mobility group box protein 1 (HMGB1) is a highly versatile, abundant, and ubiquitously expressed, non-histone chromosomal protein, which belongs to the HMGB family of proteins. These proteins form an integral part of the architectural protein repertoire to support chromatin structure in the nucleus. In the nucleus, the role of HMGB1 is attributed to its ability to bind to undamaged DNA, damaged DNA, and alternative (i.e. non-B) DNA structures with high affinity and subsequently induce bending of the DNA substrates. Due to its binding to DNA, HMGB1 has been implicated in critical biological processes, such as DNA transcription, replication, repair, and recombination. In addition to its intracellular functions, HMGB1 can also be released in the extracellular space where it elicits immunological responses. HMGB1 associates with many different molecules, including DNA, RNA, proteins, and lipopolysaccharides to modulate a variety of processes in both DNA metabolism and in innate immunity. In this review, we will focus on the implications of the interactions of HMGB1 with nucleic acids in DNA repair and immune responses. We report on the roles of HMGB1 in nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and DNA double-strand break repair (DSBR). We also report on its roles in immune responses via its potential effects on antigen receptor diversity generation [V(D)J recombination] and interactions with foreign and self-nucleic acids. HMGB1 expression is altered in a variety of cancers and immunological disorders. However, due to the diversity and complexity of the biological processes influenced by HMGB1 (and its family members), a detailed understanding of the intracellular and extracellular roles of HMGB1 in DNA damage repair and immune responses is warranted to ensure the development of effective HMGB1-related therapies.

The spectrum of APOBEC3 activity: From anti-viral agents to anti-cancer opportunities Publication date: November 2019 Source: DNA Repair, Volume 83 Author(s): Abby M. Green, Matthew D. Weitzman Abstract The APOBEC3 family of cytosine deaminases are part of the innate immune response to viral infection, but also have the capacity to damage cellular DNA. Detection of mutational signatures consistent with APOBEC3 activity, together with elevated APOBEC3 expression in cancer cells, has raised the possibility that these enzymes contribute to oncogenesis. Genome deamination by APOBEC3 enzymes also elicits DNA damage response signaling and presents therapeutic vulnerabilities for cancer cells. Here, we discuss implications of APOBEC3 activity in cancer and the potential to exploit their mutagenic activity for targeted cancer therapies.

Characterization of different DNA repair pathways in hepatic cells of Zebrafish (Danio rerio) Publication date: November 2019 Source: DNA Repair, Volume 83 Author(s): Simone Rutz Costa, Robson Rabelo Velasques, Mariana Leivas Müller Hoff, Marta Marques Souza, Juliana Zomer Sandrini Abstract The concern about DNA damage has directed efforts toward evaluating the genotoxic potential of physical and chemical agents. Since the extent of DNA damage is also related to the capacity of the organism in repairing the DNA, the advance of toxicological studies on this area depends on the characterization of the DNA repair mechanisms in the available models. The cellular zebrafish models, for example, replace mammalian cells to answer ecologically relevant questions on aquatic toxicology. So, the aim of the present study was to characterize the nucleotide excision repair (NER) and photoreactivation (PER) in two cellular models of Danio rerio liver, primary hepatocytes and ZF-L (Zebrafish Liver) cell line. We performed kinetic studies of the DNA damage levels after exposure to 6.8 J/m2 UVC using the T4-PDG modified Comet Assay, and determined the expression levels of important genes involved in NER, PER and base excision repair using RT-qPCR. It was observed that both ZF-L cell line and primary hepatocytes exhibit similar NER and PER activity. Primary hepatocytes showed similarities in the gene expression of most of the evaluated repair genes with the original tissue. These results indicate that both primary hepatocytes and ZF-L cells are useful models for toxicological studies aiming to evaluate NER and PER in hepatic cells. Moreover, the similarities in gene expression between the cellular models suggest that the ZF-L cells retain the DNA repair characteristics of the primary hepatocytes and, thus, could serve as replacement to this primary culture, reducing the use of animals in research.

Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1 Publication date: October 2019 Source: DNA Repair, Volume 82 Author(s): Milena Bazlekowa-Karaban, Paulina Prorok, Sonia Baconnais, Sabira Taipakova, Zhiger Akishev, Dominika Zembrzuska, Alexander V. Popov, Anton V. Endutkin, Regina Groisman, Alexander A. Ishchenko, Bakhyt T. Matkarimov, Amangeldy Bissenbaev, Eric Le Cam, Dmitry O. Zharkov, Barbara Tudek, Murat Saparbaev Abstract Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state. Graphical abstract

Functional profiling of nucleotide Excision repair in breast cancer Publication date: October 2019 Source: DNA Repair, Volume 82 Author(s): Anne S. Rajkumar-Calkins, Raphael Szalat, Matija Dreze, Iman Khan, Zoë Frazier, Elizaveta Reznichenkov, Mathew R. Schnorenberg, Yi-Fang Tsai, Huy Nguyen, Bose Kochupurakkal, Alan D D’Andrea, Geoffrey I Shapiro, Jean-Bernard Lazaro, Kent W Mouw Abstract Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.

A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells Publication date: October 2019 Source: DNA Repair, Volume 82 Author(s): Sandra Bosshard, Pierre-Olivier Duroy, Nicolas Mermod Abstract CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR. This dependency was influenced by the CRISPR nuclease type and the DSB-to-mutation distance, but not by the DNA sequence surrounding the DSBs or reporter cell line. We also identified elevated Mre11 and Pari, and low Rad51 expression levels as the most rate-limiting factors for HR in CHO cells. Counteracting these three bottlenecks improved precise genome editing by up to 75%. Altogether, our study provides novel insights into the complex interplay of alt-EJ and HR repair pathways, highlighting their relevance for developing improved genome editing strategies.

Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay Publication date: October 2019 Source: DNA Repair, Volume 82 Author(s): Jan Jamroskovic, Ikenna Obi, Anahita Movahedi, Karam Chand, Erik Chorell, Nasim Sabouri Abstract In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers. Graphical abstract

A novel DNA repair inhibitor, diallyl disulfide (DADS), impairs DNA resection during DNA double-strand break repair by reducing Sae2 and Exo1 levels Publication date: October 2019 Source: DNA Repair, Volume 82 Author(s): Chen-Hsin Kuo, Yann-Lii Leu, Tong-Hong Wang, Wei-Che Tseng, Chun-Hao Feng, Shu-Huei Wang, Chin-Chuan Chen Abstract Combining natural products with chemotherapy and/or radiotherapy may increase the efficacy of cancer treatment. It has been hypothesized that natural products may inhibit DNA repair and sensitize cancer cells to DNA damage-based cancer therapy. However, the molecular mechanisms underlying these activities remain unclear. In this study, we found that diallyl disulfide (DADS), an organosulfur compound, increased the sensitivity of yeast cells to DNA damage and has potential for development as an adjuvant drug for DNA damage-based cancer therapy. We induced HO endonuclease to generate a specific DNA double-strand break (DSB) by adding galactose to yeast and used this system to study how DADS affects DNA repair. In this study, we found that DADS inhibited DNA repair in single-strand annealing (SSA) system and sensitized SSA cells to a single DSB. DADS impaired DNA repair by inhibiting the protein levels of the DNA resection-related proteins Sae2 and Exo1. We also found that the recruitment of MRX and the Mec1-Ddc2 complex to a DSB was prevented by DADS. This result suggests that DADS counteracts G2/M DNA damage checkpoint activation in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Only by elucidating the molecular mechanisms by which DADS influences DNA repair will we be able to discover new adjuvant drugs to improve chemotherapy and/or radiotherapy. Graphical abstract