Snm1B/Apollo functions in the Fanconi anemia pathway in response to DNA interstrand crosslinks

Abstract

Fanconi anemia (FA) is an inherited chromosomal instability disorder characterized by childhood aplastic anemia, developmental abnormalities and cancer predisposition. One of the hallmark phenotypes of FA is cellular hypersensitivity to agents that induce DNA interstrand crosslinks (ICLs), such as mitomycin C (MMC). FA is caused by mutation in at least 14 genes which function in the resolution of ICLs during replication. The FA proteins act within the context of a protein network in coordination with multiple repair factors that function in distinct pathways. SNM1B/Apollo is a member of metallo-β-lactamase/βCASP family of nucleases and has been demonstrated to function in ICL repair. However, the relationship between SNM1B and the FA protein network is not known. In the current study, we establish that SNM1B functions epistatically to the central FA factor, FANCD2, in cellular survival after ICL damage and homology-directed repair of DNA double-strand breaks. We also demonstrate that MMC-induced chromosomal anomalies are increased in SNM1B-depleted cells, and this phenotype is not further exacerbated upon depletion of either FANCD2 or another key FA protein, FANCI. Furthermore, we find that SNM1B is required for proper localization of critical repair factors, including FANCD2, BRCA1 and RAD51, to MMC-induced subnuclear foci. Our findings demonstrate that SNM1B functions within the FA pathway during the repair of ICL damage.

A prototypical Fanconi anemia pathway in lower eukaryotes?

“…1 As ICLs can arise endogenously, cells have adapted to this threat throughout evolution by the co-ordination of several repair activities in a complex network, termed ICL repair. The first models of ICL repair emerged from studies in Escherichia coli, where the sequential action of nucleotide excision repair (NER) and homologous recombination repair (HRR) appears Fanconi anemia (FA) factor FANCD2, 27 indicating that this Pso2 ortholog functions within the FA pathway during ICL repair, and we have reported collaboration between SNM1A and XPF-ERCC1 in initiating ICL repair in replicating cells. 12 The major functional homolog of yeast Pso2 appears to be SNM1A, as expression of SNM1A in pso2 disruptant cells can partially complement ICL sensitivity and elevated ICL-associated DSBs.…”

Advances in Understanding the Complex Mechanisms of DNA Interstrand Cross-Link Repair

“…(Wang et al 2011). SNM1B in turn is epistatic with FANCD2 and FANCI (Mason and Sekiguchi 2011) and co-immunoprecipitates with FANCP/SLX4 (Salewsky et al 2012). These studies strongly implicate both nucleases in ICL repair but what their relative contributions are remains to be established.…”

Snm1B Interacts with PSF2

“…Knockdown of either Snm1B or PSF2 sensitizes cells to ICLs [2], [3], [28]. Moreover, we speculate that both proteins are likely to function in the Fanconi Anemia pathway as knockdown of Snm1B does not increase the sensitivity of FANCD2-deficient cells to MMC, and PSF2 interacts with the FANCF protein [4], [28]. Given that PSF2 localizes to the replication fork, we further speculate that perhaps this protein recruits Snm1B to forks stalled at ICLs in chromatin.…”

“…One protein involved in the repair of ICLs is Snm1B (Apollo/Dclre1B) [2], [3], [4], [5], [6], [7]. Snm1B is a 60 kDa protein belonging to the β-CASP family of proteins, which also contains Snm1A (Dclre1A) and Snm1C (Artemis/Dclre1C) [8].…”

“…Snm1C is involved in nonhomologous end joining and has a structure-specific endonuclease activity dependent upon binding DNA-PKcs [9], [15]. Snm1B is required for proper ICL repair, as knockdown of this protein leads to sensitivity of cells to ICLs [2], [3], [4], [13] and blocks the formation of double-strand breaks (DSBs) that occur as an intermediate in ICL repair [3], [16]. The enzymatic activity of Snm1B appears to be dispensable for ICL repair, although the conserved β-CASP and Metallo-β-Lactamase domains are required [3].…”