DNA Adducts of Decarbamoyl Mitomycin C Efficiently Kill Cells without Wild-Type p53 Resulting from Proteasome-Mediated Degradation of Checkpoint Protein 1

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-β-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-β-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-β stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein

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

Disruption of Mouse SNM1 Causes Increased Sensitivity to the DNA Interstrand Cross-Linking Agent Mitomycin C

DNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to nucleotide excision, recombination, and translesion DNA repair pathways in Escherichia coli, Saccharomyces cerevisiae, and mammals. We cloned and analyzed mammalian homologs of the S. cerevisiae gene SNM1 (PSO2), which is specifically involved in ICL repair. Human Snm1, a nuclear protein, was ubiquitously expressed at a very low level. We generated mouse SNM1(−/−) embryonic stem cells and showed that these cells were sensitive to mitomycin C. In contrast to S. cerevisiae snm1 mutants, they were not significantly sensitive to other ICL agents, probably due to redundancy in mammalian ICL repair and the existence of other SNM1 homologs. The sensitivity to mitomycin C was complemented by transfection of the human SNM1 cDNA and by targeting of a genomic cDNA-murine SNM1 fusion construct to the disrupted locus. We also generated mice deficient for murine SNM1. They were viable and fertile and showed no major abnormalities. However, they were sensitive to mitomycin C. The ICL sensitivity of the mammalian SNM1 mutant suggests that SNM1 function and, by implication, ICL repair are at least partially conserved between S. cerevisiae and mammals

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