Preferential repair in <i>Saccharomyces cerevisiae rad</i> mustants after induction of interstrand cross-links by 8-methoxypsoralen plus UVA

117

110

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.

Section: Discussionsupporting

confidence: 70%

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

Dronkert

Wit

Boeve

et al. 2000

117

110

“…It was also observed that the conserved domain within the Snm1 homologs represents a metallo-␤-lactamase fold, implying that these proteins contain a hydrolase activity that has been reported to encode a nuclease activity that may be involved in processing the ends of double-strand breaks (31). Since studies with yeast snm1 mutants have shown that they are not deficient in the initial incision events that occur at crosslinks but are defective in a later step required for the restoration of high-molecularweight DNA from fragmented DNA (24,27), the yeast homolog may be involved in the recognition and/or processing of double-strand breaks that occur as intermediates during interstrand crosslink repair.…”

Section: Discussionmentioning

confidence: 99%

“…Molecular analysis of snm1 mutants treated with either nitrogen mustard or psoralen plus UVA showed that incision near the sites of interstrand crosslinks proceeded normally; however, a late step in interstrand crosslink repair that is required for restoration of high-molecular-weight DNA appeared to be defective (24,27). Molecular cloning of the yeast SNM1 gene showed that it encoded a novel 76-kDa protein with a nuclear localization signal and one putative zinc finger motif (14,38).…”

mentioning

confidence: 99%

hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

Richie¹,

Peterson²,

Lü

et al. 2002

snm1 mutants of Saccharomyces cerevisiae have been shown to be specifically sensitive to DNA interstrand crosslinking agents but not sensitive to monofunctional alkylating agents, UV, or ionizing radiation. Five homologs of SNM1 have been identified in the mammalian genome and are termed SNM1, SNM1B, Artemis, ELAC2, and CPSF73. To explore the functional role of human Snm1 in response to DNA damage, we characterized the cellular distribution and dynamics of human Snm1 before and after exposure to DNAdamaging agents. Human Snm1 was found to localize to the cell nucleus in three distinct patterns. A particular cell showed diffuse nuclear staining, multiple nuclear foci, or one or two larger bodies confined to the nucleus. Upon exposure to ionizing radiation or an interstrand crosslinking agent, the number of cells exhibiting Snm1 bodies was reduced, while the population of cells with foci increased dramatically. Indirect immunofluorescence studies also indicated that the human Snm1 protein colocalized with 53BP1 before and after exposure to ionizing radiation, and a physical interaction was confirmed by coimmunoprecipitation assays. Furthermore, human Snm1 foci formed after ionizing radiation were largely coincident with foci formed by human Mre11 and to a lesser extent with those formed by BRCA1, but not with those formed by human Rad51. Finally, we mapped a region of human Snm1 of approximately 220 amino acids that was sufficient for focus formation when attached to a nuclear localization signal. Our results indicate a novel function for human Snm1 in the cellular response to double-strand breaks formed by ionizing radiation.The cellular response to DNA damage is a complex interacting network of pathways that mediate cell cycle checkpoints, DNA repair, and apoptosis. A model lesion for the investigation of these pathways has been DNA double-strand breaks, which rapidly induce cell cycle checkpoints and are repaired by a number of different pathways. In Saccharomyces cerevisiae, these lesions are preferentially repaired by a homologous recombination pathway, which requires proteins from the RAD52 epistasis group. In mammalian cells, both homologous recombination and nonhomologous recombination pathways are utilized. Extensive studies in mammalian cells have shown that complexes of DNA repair and cell cycle checkpoint proteins rapidly localize to sites of double-strand breaks induced by ionizing radiation and create foci that can be detected by immunofluorescent analyses.Two types of mutually exclusive or nonoverlapping ionizing radiation-induced foci have been observed, those containing the Rad51 protein and those containing the Mre11-Rad50-NBS1 complex (13, 25). Rad51 foci, which contain the tumor suppressor proteins BRCA1 and BRCA2, also appear during S phase in the absence of exogenous induction of DNA damage (5,29,(43)(44)(45). Mre11-Rad50-NBS1 foci can be detected as early as 10 min after irradiation and are clearly present at sites of DNA breaks while DNA repair is ongoing (25,28,33). These foci also colocal...

“…Outside the SNM1 domain, the sequences of the yeast and human proteins are different. The function of yeast Snm1 remains largely unresolved, although several studies have indicated that it is involved in repairing double-strand breaks (DSBs) resulting from processing of interstrand cross-links (31,35,40). Artemis was originally identified molecularly as deficient in a human radiosensitive severe combined immunodeficiency syndrome (RS-SCID) (41), which is characterized by a defect in V(D)J recombination resulting in premature arrest of both B-and T-cell maturation.…”

mentioning

confidence: 99%

Artemis Is a Phosphorylation Target of ATM and ATR and Is Involved in the G₂/M DNA Damage Checkpoint Response

Zhang¹,

Succi²,

Feng³

et al. 2004

106

104

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemisdepleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G 2 /M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.Artemis is a member of the SNM1/PSO2 gene family, the archetypical member of which was identified in budding yeast (Saccharomyces cerevisiae) as a factor required for efficient DNA interstrand cross-link repair (23, 53). Members of this family, which in humans also include SNM1, SNM1B, ELAC2, and CPSF73 (15,27,57), share a region of homology termed the SNM1 domain, which contains a metallo-␤-lactamase fold and an appended ␤-CASP (for metallo-␤-lactamase-associated CPSF Artemis SNM1/PSO2) domain that is a predicted nucleic acid binding motif (7,41). Outside the SNM1 domain, the sequences of the yeast and human proteins are different. The function of yeast Snm1 remains largely unresolved, although several studies have indicated that it is involved in repairing double-strand breaks (DSBs) resulting from processing of interstrand cross-links (31,35,40). Artemis was originally identified molecularly as deficient in a human radiosensitive severe combined immunodeficiency syndrome (RS-SCID) (41), which is characterized by a defect in V(D)J recombination resulting in premature arrest of both B-and T-cell maturation. In addition, patient cell lines exhibited greater sensitivity to ionizing radiation (IR) than normal cells (9, 42, 44). RS-SCID resembles murine SCID caused by defects in DNA-PK, a protein complex involved in both V(D)J recombination and repair of DSBs via the nonhomologous end-joining (NHEJ) pathway. These findings have also been confirmed in a mouse model in which the Artemis gene was disrupted by gene targeting (51). Biochemical studies of Artemis have shown that it possesses a 5Ј33Ј exonucleolytic activity on single-stranded DNA, and when complexed with DNA-PKcs, it acquires endonucleolytic activity on 5Ј and 3Ј overhangs and the ability to open DNA hairpins (34). This latter activity is consistent with the observed defect in coding joint fo...