DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation - PubMed (original) (raw)

Masamichi Ishiai et al. Mol Cell Biol. 2004 Dec.

Abstract

The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-beta-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.

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Figures

FIG. 1.

FIG. 1.

SNM1 family genes in vertebrates. (A) Schematic diagrams of SNM1A family. Yeast (y) SNM1 and hSNM1 family proteins are shown. Numbers in the SNM1 domain indicate percentages of identity to the yeast SNM1 domain. (B to D) Targeted disruption of chicken SNM1A (B), SNM1B (C), and SNM1C (D) loci in DT40 cells. Schematic representations of part of each loci, the gene targeting constructs, the configuration of targeted allele, and results of the Southern blot analysis and RT-PCR analysis are shown. White boxes indicate the positions of exons that were disrupted. B, BamHI; H, HindIII. Southern blot analysis was carried out with genomic DNA digested by BamHI (SNM1A and SNM1C) or HindIII (SNM1B) from cells with the indicated genotypes by use of flanking probes. mRNA expression of each disrupted gene or control (RAD51) in wild-type and mutant DT40 cells was analyzed by RT-PCR. (E) Nuclear localization patterns of EGFP-fused SNM1 family proteins in complemented DT40 mutant cells. WT, wild type.

FIG. 2.

FIG. 2.

Sensitivities of wild-type and SNM1 family-deficient DT40 cells to DNA-damaging agents. The fractions of the surviving colonies after treatment compared to the nontreated control of the same genotype are shown as percent survival. (A to C) Survival of snm1a (A), snm1b (B), and snm1c (C) mutant cells compared to wild-type (WT) and complemented control cells after continuous exposure to cisplatin. (D and E) Survival of snm1a, snm1b, and WT cells after 1 h of exposure to MMC (D) or IR (E). (F) Survival of snm1c, ligase4, and WT cells and _SNM1C_-complemented control cells after IR. The data shown are means ± standard deviations of results for at least three separate experiments.

FIG. 3.

FIG. 3.

Epistasis analyses between SNM1A and the indicated repair pathways. (A) Generation of an snm1a/xrcc3 double disruptant from conditional xrcc3 cells was done as described in Materials and Methods. (B to E) Survival of SNM1A double mutants with XRCC3 (B), RAD18 (C), FANCC (D), and SNM1B (E) compared to that of the wild type (WT) and corresponding single mutants after continuous exposure to cisplatin. Two clones of each double mutant were included in the analyses. The data shown are means ± standard deviations of results for at least three separate experiments.

FIG. 4.

FIG. 4.

Mutational analysis of SNM1A. (A) Schematic representations of SNM1A point mutants. Cellular distributions of each construct determined by transient transfections of HeLa cells (E) and complementation data (C) are summarized. (B) Alignment of conserved regions in the SNM1 domain of SNM1 family proteins. The amino acid sequences of the SNM1 domain of human (Hs), mouse (Mm), chicken (Gd), and yeast (Sc) SNM1 family gene products were aligned. Amino acids that were identical across 10 proteins or 6 to 9 proteins are indicated by black or grey shading, respectively. Arrowheads indicate the mutated amino acid residues (D838 and H994 in hSNM1A) in this study. (C) Survival of snm1a cells stably expressing EGFP-hSNM1A mutants in the presence of cisplatin. Fluorescence-activated cell sorting analyses of wild-type and mutant EGFP-SNM1A expression are shown in the lower panels. Nontransfected snm1a cells were used as a negative control, and their fluorescence levels are shown as grey lines. (D) Localization of EGFP-SNM1A mutants transiently expressed in HeLa cells. Cells were examined with a confocal laser microscope.

FIG. 5.

FIG. 5.

Interaction of hSNM1A and hPIAS1. (A)Yeast two-hybrid assay. Yeast cells were cotransformed with the bait plasmid containing either hSNM1AΔ1-554 or its derivatives (with the D838N or H994A mutation) and the prey plasmid containing hPIAS1. Empty bait plasmid was used for control. _β_-Galactosidase activity was determined by a liquid culture assay and calculated in Miller units. (B and C) 293T cells were cotransfected with the indicated expression plasmids. Ni-NTA resin pull-down analysis was carried out as described in Materials and Methods. Samples were analyzed by Western blotting with anti-GFP or anti-His antibodies.

FIG. 6.

FIG. 6.

Colocalization of SNM1A and PIAS1 in HeLa cells. (A) Localization of transiently transfected EGFP-hSNM1A or its mutants (with the D838N or H994A mutation) and His/Max-hPIAS1. PIAS1 was detected by use of anti-Xpress and Alexa fluor 594-conjugated secondary antibody. Images were captured by confocal microscopy. (B) Localization of His/Max-SNM1A and EGFP-PIAS1 were analyzed as described for panel A.

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References

    1. Adachi, N., T. Ishino, Y. Ishii, S. Takeda, and H. Koyama. 2001. DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: implications for DNA double-strand break repair. Proc. Natl. Acad. Sci. USA 98:12109-12113. - PMC - PubMed
    1. Brendel, M., D. Bonatto, M. Strauss, L. F. Revers, C. Pungartnik, J. Saffi, and J. A. P. Henriques. 2003. Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae. Mutat. Res. 544:179-193. - PubMed
    1. Callebaut, I., D. Moshous, J.-P. Mornon, and J.-P. de Villartay. 2002. Metallo-β-lactamase fold within nucleic acids processing enzymes: the β-CASP family. Nucleic Acids Res. 30:3592-3601. - PMC - PubMed
    1. D'Andrea, A. D., and M. Grompe. 2003. The Fanconi anaemia/BRCA pathway. Nat. Rev. Cancer 3:23-34. - PubMed
    1. De Silva, I. U., P. J. McHugh, P. H. Clingen, and J. A. Hartley. 2000. Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Mol. Cell. Biol. 20:7980-7990. - PubMed

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