Visualizing replication fork encounters with DNA interstrand crosslinks.

Localization of αII Spectrin to Telomeres After DNA Interstrand Cross-Link Damage Is Deficient In Fanconi Anemia Group A Cells, Which May Lead to Telomere Dysfunction.

The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5-2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro.

Inhibition of CDC7 with Sgr-2921 in AML Models Results in Enhanced DNA Damage and Anti-Leukemic Activity As Monotherapy and in Combination with Standard of Care Agents

A hallmark of the cancer prone genetic disorder, Fanconi anemia (FA), is genomic instability characterized by both chromosome instability and defects in DNA interstrand crosslink (ICL) repair. The chromosome instability, in particular after exposure to ICLs, suggests telomere dysfunction. We have demonstrated that the structural protein, nonerythroid α spectrin (αIISp), is present in the nucleus of normal human cells where it is critical in repair of DNA ICLs and maintenance of chromosome stability and that in FA cells levels of αIISp are reduced due to its increased cleavage by μ-calpain, which correlate with reduced ICL repair and increased chromosomal instability. The goal of this study was to determine whether αIISp is important for telomere function after DNA ICL damage and whether telomere dysfunction in FA cells after damage can be corrected when levels of αIISp are restored to normal. Normal and FA complementation group A (FA-A) lymphoblastoid cells were treated with a DNA ICL agent, 8-methoxypsoralen plus UVA light or mitomycin C and studied using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with anti-α-spectrin and either a telomeric probe (PNA) or antibodies against two telomeric proteins, TRF1 or TRF2. The results showed that 47-50% of αIISp foci colocalized with the PNA probe, TRF1 or TRF2 in telomeres in ICL damaged but not undamaged normal cells and that this colocalization was specific for the S phase. This indicated that αIISp may play an important role in telomere function after ICLs and this view was strengthened by data showing that αIISp has markedly enhanced binding for TRF1 and TFR2 after ICL damage. IF-FISH showed that the ICL repair protein, XPF, also had enhanced association with telomeres after ICL formation. In FA-A cells, no localization of XPF to telomeres was observed; however, after αIISp levels were returned to normal by siRNA knockdown of μ-calpain, αIISp as well as XPF associated with telomeres after ICLs. The chromosomal aberrations (i.e., breaks and end fusions) observed in FA-A cells after ICL damage were also corrected. The present results suggest that, after ICL damage, αIISp associates with telomeres in S phase and this association may be important in repair of ICLs in replicating telomeres. In FA-A cells, failure of localization of αIISp and XPF to telomeres after ICL damage could lead to unrepaired ICLs, telomere dysfunction and production of the chromosomal aberrations observed in these cells. These studies show that αIISp may play an important role in telomere function after DNA damage and during telomere replication. Correction of the deficiency in αIISp in FA cells by knocking down μ-calpain may be significant in reversing telomere dysfunction and chromosomal instability after ICL damage and could have important therapeutic relevance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3924. doi:10.1158/1538-7445.AM2011-3924

Summary:Cells often utilize multiple pathways to repair the same DNA lesion, and pathway choice has profound implications for the fidelity of genome maintenance. DNA interstrand cross-links (ICLs) block DNA replication and transcription by covalently linking the two strands of DNA, and the cytotoxicity of ICLs is exploited for chemotherapy. In Xenopus egg extracts, replication fork collision with ICLs initiates two distinct repair pathways. The NEIL3 glycosylase can cleave the cross-link1, but if this fails, the Fanconi anemia (FA) proteins incise the phosphodiester backbone surrounding the ICL, generating a double-strand break (DSB) intermediate that is repaired by homologous recombination2. How the simpler NEIL3 pathway is prioritized over the FA pathway, which can cause genomic rearrangements, is unknown. Here, we show that the E3 ubiquitin ligase TRAIP regulates both pathways. TRAIP appears to associate with the leading edge of the replisome, ubiquitylating any protein in the replisome’s path, including the replicative DNA helicase CMG (CDC45-MCM2-7-GINS) when two replisomes converge at an ICL. In this setting, short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for CMG unloading by the p97 ATPase, enabling the FA pathway. Our results identify TRAIP as a master regulator of replisome stability and ICL repair pathway choice.

The Role of Active Oxygen (102 and 0·2-) Induced by Crude Coal Tar and Its Ingredients Used in Photochemotheraphy of Skin Diseases

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the "checkpoint Rad" family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G(2) arrest following ICL induction. In cells able to bypass the G(2) block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G(2) arrest-deficient mutants. This likely reflects the fraction of cells escaping the G(2) damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Replication-Independent Repair of DNA Interstrand Crosslinks

DNA interstrand crosslinks (ICLs) formed by antitumor agents, such as cisplatin or mitomycin C, are highly cytotoxic DNA lesions. Their repair is believed to be triggered primarily by the stalling of replication forks at ICLs in S-phase. There is, however, increasing evidence that ICL repair can also occur independently of replication. Using a reporter assay, we describe a pathway for the repair of cisplatin ICLs that depends on transcription-coupled nucleotide excision repair protein CSB, the general nucleotide excision repair factors XPA, XPF and XPG, but not the global genome nucleotide excision repair factor XPC. In this pathway, Rev1 and Polζ are involved in the error-free bypass of cisplatin ICLs. The requirement for CSB, Rev1 or Polζ is specific for the repair of ICLs, as the repair of cisplatin intrastrand crosslinks does not require these genes under identical conditions. We directly show that this pathway contributes to the removal of ICLs outside of S-phase. Finally, our studies reveal that defects in replication- and transcription-dependent pathways are additive in terms of cellular sensitivity to treatment with cisplatin or mitomycin C. We conclude that transcription- and replication-dependent pathways contribute to cellular survival following treatment with crosslinking agents.

MCM2-7 and FANCD2 Are Required for RAD51 Function in Homologous Recombination and ICL Repair

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment

Interstrand DNA crosslinks (ICLs) are a highly toxic form of DNA damage. ICL repair in both eukaryotes and bacteria involves unhooking of the two strands by specialized DNA glycosylases. We recently established that the human pathogen Acinetobacter baumannii contains an ICL glycosylase (AlkX) that facilitates pathogenesis and protects the bacteria from DNA damage and acid stress. However, the physical basis for glycosylase-catalyzed ICL unhooking is unknown. Here, we describe a crystal structure of AlkX bound to DNA representing a product of the ICL unhooking reaction. Mutational analysis of ICL unhooking in vitro and A. baumannii sensitivity to the crosslinking agent mechlorethamine enabled identification of several AlkX motifs critical for ICL repair. We also found that a genetic variant from an antibiotic-resistant strain of the human pathogen Salmonella enterica significantly reduced AlkX activity in vitro and increased A. baumannii sensitivity to DNA crosslinking. This work provides a structural basis for how bacterial ICL glycosylases recognize and repair DNA adducts and contributes additional evidence that ICL repair is important for fitness of human pathogens.

DNA interstrand crosslinks (ICLs) are extremely cytotoxic lesions that block essential cellular processes, such as replication and transcription. Crosslinking agents are widely used in cancer chemotherapy and form an array of structurally diverse ICLs. Despite the clinical success of these agents, resistance of tumors to crosslinking agents, for example, through repair of these lesions by the cellular machinery remains a problem. We have previously reported the synthesis of site-specific ICLs mimicking those formed by nitrogen mustards to facilitate the studies of cellular responses to ICL formation. Here we extend these efforts and report the synthesis of structurally diverse major groove ICLs that induce severe, little or no distortion in the DNA. Our approach employs the incorporation of aldehyde precursors of different lengths into complementary strands and ICL formation using a double reductive amination with a variety of amines. Our studies provide insight into the structure and reactivity parameters of ICL formation by double reductive amination and yield a set of diverse ICLs that will be invaluable for exploring structure–activity relationships in ICL repair.