The human DNA glycosylases NEIL1 and NEIL3 excise psoralen-induced DNA-DNA cross-links in a four-stranded DNA structure

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death

Xpf and Not the Fanconi Anaemia Proteins or Rev3 Accounts for the Extreme Resistance to Cisplatin in Dictyostelium discoideum

Organisms like Dictyostelium discoideum, often referred to as DNA damage “extremophiles”, can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other “extremophiles” can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage–resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents

The analysis of DNA interstrand cross-links and their repair by the modified comet assay

DNA interstrand cross-links (ICLs) are an extremely toxic form of DNA damage that cells experience upon exposure to natural metabolites. Moreover, ICLs are cytotoxic lesions produced by a range of clinically important anticancer agents. Therefore, improving our understanding of ICL induction and processing has important implications in biology and medicine. The sensitive detection of ICLs in mammalian cells is challenging but has been aided by the development of a modified form of the single-cell gel electrophoresis (SCGE) assay, also known as the “comet assay.” Here we describe this method and how it can be used to sensitively monitor the induction and removal of ICLs in single mammalian cells

Sister chromatid exchanges occur in G2-irradiated cells

DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or misrepaired DSBs can lead to chromosomal aberrations and cell death. The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. Following S-phase-dependent genotoxins, HR events give rise to sister chromatid exchanges (SCEs), which can be detected cytogenetically in mitosis. Here, we describe that HR occurring in G2-irradiated cells also generates SCEs in ∼50% of HR events. Since HR of IR-induced DSBs in G2 is a slow process, SCE formation in G2-irradiated cells requires several hours. During this time, irradiated S-phase cells can also reach mitosis, which has contributed to the widely held belief that SCEs form only during S phase. We describe procedures to measure SCEs exclusively in G2-irradiated cells and provide evidence that following IR cells do not need to progress through S phase in order to form SCEs

Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widely used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. These findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus

DdrA, DdrD, and PprA: Components of UV and Mitomycin C Resistance in Deinococcus radiodurans R1

Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species’ resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm(-2), the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species