DNA Cross-Link Repair Protein SNM1A Interacts with PIAS1 in Nuclear Focus Formation

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-␤-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.DNA interstrand cross-links (ICLs) covalently bind the two complementary strands of the double helix of DNA. They severely impair fundamental processes of DNA metabolism such as transcription or replication, leading to cell death if they are left unrepaired. Current protocols for cancer chemotherapy often include ICL-inducing agents, i.e., mitomycin C (MMC) or cisplatin, for effective killing of malignant cells (reviewed in references 7 and 21).The molecular mechanism of ICL repair is still poorly understood. In the yeast Saccharomyces cerevisiae, three distinct pathways, including nucleotide excision repair (NER), homologous recombination (HR), and translesion synthesis (TLS), participate in ICL repair (7,20). In addition, yeast SNM1 (also known as PSO2) specifically functions in ICL repair without apparently participating directly in any of these pathways (2, 9). During the repair process, NER mediates incisions on both sides of the cross-linked DNA (sometimes termed "unhooking") (5), and double-strand breaks (DSBs), which are probably associated with the collapse of a replication fork, are formed. The DSBs are then repaired by HR mechanisms. The interdependence between unhooking and formation of DSBs remains unclear (7). Yeast snm1 mutants are proficient in unhooking but are unable to process DSB intermediates (7,19,40).In mammalian cells, the situation is even more complex.Among NER factors, XPF/ERCC1 endonuclease appears to be particularly important for the unhooking of ICLs (5,21,26,32). Hamster mutant cells lacking the RAD51 paralog XRCC2 or XRCC3 display extreme sensitivity to ICLs, indicating an important role of HR in mammalian ICL repair (17). Cells from Fanconi anemia (FA) patients are also highly sensitive to ICL reagents (33), but the role of FA proteins in ICL repair is still unclear (4). Furthermore, there are three homologs of SNM1 (referred to as SNM1A, S...

hSnm1B Is a Novel Telomere-associated Protein

“…The localization of these fluorescent proteins was then compared with that of endogenous TRF2, as measured by immunofluorescence. As reported previously, both YFP-FLAG-hSnm1A and GFP-hSnm1B localized to discrete punctate nuclear bodies (28). However, only GFP-hSnm1B co-localized with endogenous TRF2 at the telomere (Fig.…”

“…Knock-out of Snm1B in chicken cells and small interfering RNA against Snm1B in human cells both result in mild sensitivity to interstrand cross-linking agents (28,29). Knockdown of Snm1B by small interfering RNA induces an increase in aberrant metaphase morphology in human cells (29).…”

Endogenous hSNM1B/Apollo interacts with TRF2 and stimulates ATM in response to ionizing radiation

“…As shown above, the anti-hSNM1B antibodies were able to detect hSNM1B in IF experiments which allowed us to determine whether endogenous hSNM1B localizes to telomeres, as suggested by the yeast-two-hybrid and co-IP results and previously published results on ectopic overexpressed hSNM1B [12][13][14][15]. Double staining of hSNM1B and either of the telomere markers, TRF1 or TRF2, demonstrated a high degree of co-localization of these proteins ( Figure 2C) and showing, for the first time, that the majority of endogenous hSNM1B foci are localized at telomers.…”

Snm1B/Apollo mediates replication fork collapse and S Phase checkpoint activation in response to DNA interstrand cross-links

“…Snm1A has been shown to colocalize with Mre11 foci after exposure of cells to IR or ICL-inducing agents, and to interact with the checkpoint protein 53BP1 (Richie et al, 2002). However, mammalian SNM1A-deficient cells exhibit no hypersensitivity to IR and only a modest hypersensitivity to interstrand cross-linking agents (Dronkert et al, 2000;Ahkter et al, 2005), although sensitivity to cisplatin has been observed in chicken DT40 cells (Ishiai et al, 2004;Nojima et al, 2005). Intriguingly, Snm1A-deficient mouse embryonic fibroblasts are highly sensitive to spindle poisons such as nocodazole and taxol, and Snm1A has been shown to be involved in an early mitotic checkpoint pathway in response to these drugs (Akhter et al, 2004).…”

“…In chicken and mammalian cells three orthologues of SNM1 have been identified that are involved in the cellular response to genotoxic agents (Dronkert et al, 2000;Ishiai et al, 2004). These genes include SNM1A, SNM1B/Apollo and Artemis.…”