RAD18-dependent Recruitment of SNM1A to DNA Repair Complexes by a Ubiquitin-binding Zinc Finger

Abstract

SNM1A is a member of the SNM1 family of nucleases required for cellular processing of interstrand DNA crosslinks (ICLs). Little is known about the molecular function of SNM1A, in terms of its recruitment to ICL lesions or its DNA damage processing activity. Here we show that SNM1A contains a functional PIP box (PCNA-interacting protein box) and a UBZ (ubiquitin binding zinc finger), required for assembly of SNM1A into nuclear focus. Moreover, RAD18-dependent monoubiquitination of PCNA is required for Mitomycin C and Ultraviolet Light inducible SNM1A nuclear focus assembly. Taken together, our results identify a novel RAD18-PCNA(Ub)-SNM1A pathway required for nuclear focus formation and ICL resistance.The repair of DNA interstrand crosslinks (ICLs) 4 in mammalian cells requires the coordinate activity of three discrete pathways, including nucleotide excision repair (NER), homologous recombination (HR), and translesion DNA synthesis (TLS) (1). In addition, yeast SNM1 (also known as Pso2) is required for ICL repair and appears to function independently of these three pathways. Pso2 is a member of the ␤-CASP metallo-␤-lactamase family of nucleases (2). Human cells have three homologs of SNM1 (referred to as SNM1A, SNM1B/Apollo, and SNM1C/ Artemis), and their relative contribution to ICL repair remains unknown.Recent studies have elucidated some of the functions of the SNM1A protein. First, human SNM1A, unlike SNM1B and SNM1C, can rescue the crosslinker hypersensitivity of a yeast Pso2 mutant, suggesting that SNM1A is the homologue of the yeast protein (3). Second, a murine knock-out of SNM1A is hypersensitive to MMC, but not to ionizing radiation (IR) (4 -6). Third, in response to DNA damage, SNM1A assembles in subnuclear foci (7), further supporting its role in the DNA damage response. Fourth, SNM1A can function as a DNA exonuclease in vitro, suggesting that it may have a direct role in excising DNA ICLs (3,8).DNA repair proteins are often recruited to specific sites of DNA damage through protein-protein interaction domains. For example, the FHA domain of NBS1 and the BRCT domains of BRCA1 are required for recruitment of these DNA repair proteins to complexes containing phosphopeptide motifs (9). Ubiquitin binding motifs (UBMs) and ubiquitin-binding zinc fingers (UBZs) are found in some DNA repair proteins and can target these proteins to monoubiquitinated complexes (10 -12). The UBZ motif of Pol for example, can recruit this protein to monoubiquitinated PCNA. However, the molecular mechanism by which SNM1A is recruited to sites of ICL repair is currently unknown (7).In this study, we propose a mechanism for the damage-inducible recruitment of SNM1A to sites of DNA damage. We identified a conserved UBZ domain in SNM1A, which is functional for monoubiquitin binding. DNA damage, by MMC or ultraviolet light, activated the UBZ-dependent recruitment of SNM1A into nuclear foci. Interestingly, this recruitment was RAD18 dependent, suggesting that RAD18 monoubiquitinates PCNA, leading to the direct interaction of UBZ-SNM...

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