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DNA interstrand cross-links, such as those formed by psoralen-UVA irradiation, are highly toxic lesions in both humans and bacteria, with a single lesion being lethal in Escherichia coli. Despite the lack of effective repair, human cancers and bacteria can develop resistance to cross-linking treatments, although the mechanisms of resistance remain poorly defined. Here, we subjected E. coli to repeated psoralen-UVA exposure to isolate three independently derived strains that were >10,000-fold more resistant to this treatment than the parental strain. Analysis of these strains identified gain-of-function mutations in the transcriptional regulator AcrR and the alpha subunit of RNA polymerase that together could account for the resistance of these strains. Resistance conferred by the AcrR mutation is mediated at least in part through the regulation of the AcrAB-TolC efflux pump. Resistance via mutations in the alpha subunit of RNA polymerase occurs through a still-uncharacterized mechanism that has an additive effect with mutations in AcrR. Both acrR and rpoA mutations reduced cross-link formation in vivo. We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. IMPORTANCE Psoralen DNA interstrand cross-links are highly toxic lesions with antimicrobial and anticancer properties. Despite the lack of effective mechanisms for repair, cells can become resistant to cross-linking agents through mechanisms that remain poorly defined. We derived resistant mutants and identified that two gain-of-function mutations in AcrR and the alpha subunit of RNA polymerase confer high levels of resistance to E. coli treated with psoralen-UVA. Resistance conferred by AcrR mutations occurs through regulation of the AcrAB-TolC efflux pump, has an additive effect with RNA polymerase mutations, acts by reducing the formation of cross-links in vivo, and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell.

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DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community. • The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand. • Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures. • The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues.

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DNA interstrand cross-links (ICLs) are lesions with a covalent bond formed between DNA strands. ICLs are extremely toxic to cells because they prevent the separation of the two strands, which are necessary for the genetic interpretation of DNA. ICLs are repaired via Fanconi anemia and replication-independent pathways. The formation of so-called unhooked repair intermediates via a dual strand incision flanking the ICL site on one strand is an essential step in nearly all ICL repair pathways. Recently, ICLs derived from endogenous sources, such as those from ubiquitous DNA lesions, abasic (AP) sites, have emerged as an important class of ICLs. Despite the earlier efforts in preparing AP-ICLs in high yield using nucleotide analogs, little information is available for preparing AP-ICL unhooked intermediates with varying lengths of overhangs. In this study, we devise a simple approach to prepare model ICL unhooked intermediates derived from AP sites. We exploited the alkaline lability of ribonucleotides (rNMPs) and the high cross-linking efficiency between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination. We designed chimeric DNA/RNA substrates with rNMPs flanking the cross-linking residue (2-aminopurine) to facilitate subsequent strand cleavage under our optimized conditions. Mass spectrometric analysis and primer extension assays confirmed the structures of ICL substrates. The method is straightforward, requires no synthetic chemistry expertise, and should be broadly accessible to all researchers in the DNA repair community. For step-by-step descriptions of the method, please refer to the companion manuscript in MethodsX.

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DNA interstrand cross-links (ICLs) are essential for the antitumor activity of chloroethylnitrosoureas (CENUs). Commonly, CENUs resistance is mainly considered to be associated with O6-methylguanine-DNA methyltransferase (MGMT) within tumors. Bypassing the MGMT-mediated resistance, to our knowledge, herein, we first utilized a novel glycolytic inhibitor, 3-bromopyruvate (3-BrPA), to increase the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to human glioma cells based on the hypothesis that blocking energy metabolism renders tumor cells more sensitive to chemotherapy. We found 3-BrPA significantly increased the cell killing by BCNU in human glioma SF763 and SF126 cell lines. Significantly decreased levels of extracellular lactate, cellular ATP and glutathione (GSH) were observed after 3-BrPA treatment, and the effects were more remarkable with 3-BrPA in combination with BCNU. Considering that the role of ATP and GSH in drug efflux, DNA damage repair and drug inactivation, we determined the effect of 3-BrPA on the formation of dG-dC ICLs induced by BCNU using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As expected, the levels of lethal dG-dC ICLs induced by BCNU were obviously enhanced after 3-BrPA pretreatment. Based on these results, 3-BrPA and related glycolytic inhibitors may be promising to enhance the cell killing effect and reverse the clinical chemoresistance of CENUs and related antitumor agents.

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Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow failure (BMF), congenital defects, inability to repair DNA interstrand cross-links (ICLs), and cancer predisposition. FA presents two seemingly opposite characteristics: (a) massive cell death of the hematopoietic stem and progenitor cell (HSPC) compartment due to extensive genomic instability, leading to BMF, and (b) uncontrolled cell proliferation leading to FA-associated malignancies. The canonical function of the FA proteins is to collaborate with several other DNA repair proteins to eliminate clastogenic (chromosome-breaking) effects of DNA ICLs. Recent discoveries reveal that the FA pathway functions in a critical tumor-suppressor network to preserve genomic integrity by stabilizing replication forks, mitigating replication stress, and regulating cytokinesis. Homozygous germline mutations (biallelic) in 22 FANC genes cause FA, whereas heterozygous germline mutations in some of the FANC genes (monoallelic), such as BRCA1 and BRCA2, do not cause FA but significantly increase cancer susceptibility sporadically in the general population. In this review, we discuss our current understanding of the functions of the FA pathway in the maintenance of genomic stability, and we present an overview of the prevalence and clinical relevance of somatic mutations in FA genes.

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Chloroethylnitrosoureas (CENUs) are an important type of alkylating agent employed in the clinical treatment of cancer. However, the anticancer efficacy of CENUs is greatly decreased by a DNA repairing enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), by preventing the formation of interstrand cross-links (ICLs). In this study, a combi-nitrosourea prodrug, namely, N-(2-chloroethyl)-N'-2-(O6-benzyl-9-guanine)ethyl-N-nitrosourea (BGCNU), which possesses an O6-benzylguanine (O6-BG) derivative and CENU pharmacophores simultaneously, was synthesized and evaluated for its ability to induce ICLs. The target compound is markedly more cytotoxic in human glioma cells than the clinically used CENU chemotherapies ACNU, BCNU, and their respective combinations with O6-BG. In the AGT-proficient cells, significantly higher levels of DNA ICLs were observed in the groups treated by BGCNU than those by ACNU and BCNU, which indicated that the activity of AGT was effectively inhibited by the O6-BG derivatives released from BGCNU.

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Non-erythroid alpha spectrin (αIISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. αIISp's interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear αIISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maintenance of genomic stability following ICL damage to DNA. We have proposed that αIISp acts as a scaffold aiding to recruit repair proteins to sites of damage. This involvement of αIISp in ICL repair and telomere maintenance after ICL damage represents new and critical functions for αIISp. These studies have led to development of a model for the role of αIISp in DNA ICL repair. They have been aided by examination of cells from patients with Fanconi anemia (FA), a repair-deficient genetic disorder in which a deficiency in αIISp leads to defective ICL repair in genomic and telomeric DNA, telomere dysfunction, and chromosome instability following DNA ICL damage. We have shown that loss of αIISp in FA cells is due to increased breakdown by the protease, µ-calpain. Importantly, we have demonstrated that this deficiency can be corrected by knockdown of µ-calpain and restoring αIISp levels to normal. This corrects a number of the phenotypic deficiencies in FA after ICL damage. These studies suggest a new and unexplored direction for therapeutically restoring genomic stability in FA cells and for correcting numerous phenotypic deficiencies occurring after ICL damage. Developing a more in-depth understanding of the importance of the interaction of αIISp with other nuclear proteins could significantly enhance our knowledge of the consequences of loss of αIISp on critical nuclear processes.

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Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3' untranslated region of the gene. miR146a-induced downregulation of FANCM was associated with inhibition of FANCD2 monoubiquitination, reduced DNA homologous recombination repair and checkpoint response, failed recovery from replication stress, and increased cellular sensitivity to cisplatin. These phenotypes were recapitulated when miR146a expression was induced by overexpressing the NF-κB subunit p65/RelA or Helicobacter pylori infection in a human gastric cell line; the phenotypes were effectively reversed with an anti-miR146a antagomir. These results suggest that undesired inflammation events caused by a pathogen or over-induction of miR146a can impair genome integrity via suppression of FANCM.

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Nonerythroid α spectrin (αIISp) and the Fanconi anemia (FA) protein, FANCD2, play critical roles in DNA interstrand cross-link (ICL) repair during S phase. Both are needed for recruitment of repair proteins, such as XPF, to sites of damage and repair of ICLs. However, the relationship between them in ICL repair and whether αIISp is involved in FANCD2's function in repair is unclear. The present studies show that, after ICL formation, FANCD2 disassociates from αIISp and localizes, before αIISp, at sites of damage in nuclear foci. αIISp and FANCD2 foci do not co-localize, in contrast to our previous finding that αIISp and the ICL repair protein, XPF, co-localize and follow a similar time course for formation. Knock-down of αIISp has no effect on monoubiquitination of FANCD2 (FANCD2-Ub) or its localization to chromatin or foci, though it leads to decreased ICL repair. Studies using cells from FA patients, defective in ICL repair and αIISp, have elucidated an important role for αIISp in the function of non-Ub FANCD2. In FA complementation group A (FA-A) cells, in which FANCD2 is not monoubiquitinated and does not form damage-induced foci, we demonstrate that restoration of αIISp levels to normal, by knocking down the protease µ-calpain, leads to formation of non-Ub FANCD2 foci after ICL damage. Since restoration of αIISp levels in FA-A cells restores DNA repair and cell survival, we propose that αIISp is critical for recruitment of non-Ub FANCD2 to sites of damage, which has an important role in the repair response and ICL repair.

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Nonerythroid alpha spectrin (αIISp) interacts in the nucleus with an array of different proteins indicating its involvement in a number of diverse functions. However, the significance of these interactions and their functional importance has been a relatively unexplored area. The best documented role of nuclear αIISp is in DNA repair where it is critical for repair of DNA interstrand cross-links (ICLs), acting as a scaffold recruiting proteins to sites of damage in genomic and telomeric DNA. A deficiency in αIISp can importantly impact DNA ICL repair as is seen in cells from patients with the genetic disorder, Fanconi anemia (FA), where loss of αIISp leads to not only defects in repair of both genomic and telomeric DNA but also to telomere dysfunction and chromosome instability. This previously unexplored link between αIISp and telomere function is important in developing an understanding of maintenance of genomic stability after ICL damage. In FA cells, these defects in chromosome instability after ICL damage can be corrected when levels of αIISp are returned to normal by knocking down µ-calpain, a protease which cleaves αIISp. These studies suggest a new direction for correcting a number of the phenotypic defects in FA and could serve as a basis for therapeutic intervention. More in depth, examination of the interactions of αIISp with other proteins in the nucleus is of major importance in development of insights into the interacting key elements involved in the diverse processes occurring in the nucleus and the consequences loss of αIISp has on them.

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Eukaryotic cells have developed mechanisms to prevent genomic instability, such as DNA damage detection and repair, control of cell cycle progression and cell death induction. The bifunctional compound furocumarin 8-methoxypsoralen (8-MOP) is widely used in the treatment of various inflammatory skin diseases. In this review, we summarize recent data about the role of chromatin remodeling in the repair of DNA damage induced by treatment with 8-methoxypsoralen plus UVA (8-MOP+UVA), focusing on repair proteins in budding yeast Saccharomyces cerevisiae, an established model system for studying DNA repair pathways. The interstrand crosslinks (ICL) formed by the 8-MOP+UVA treatment are detrimental lesions that can block transcription and replication, leading to cell death if not repaired. Current data show the involvement of different pathways in ICL processing, such as nucleotide excision repair (NER), base excision repair (BER), translesion repair (TLS) and double-strand break repair. 8-MOP+UVA treatment in yeast enhances the expression of genes involved in the DNA damage response, double strand break repair by homologous replication, as well as genes related to cell cycle regulation. Moreover, alterations in the expression of subtelomeric genes and genes related to chromatin remodeling are consistent with structural modifications of chromatin relevant to DNA repair. Taken together, these findings indicate a specific profile in 8-MOP+UVA responses related to chromatin remodeling and DNA repair.