Abstract
The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells.
Highlights
The XPF-ERCC1 complex is a highly conserved heterodimeric structure-specific endonuclease (SSE) composed of the XPF catalytic subunit and the ERCC1 DNA binding subunit that is involved in DNA repair and maintenance of chromosome stability (Dehé and Gaillard, 2017; Faridounnia et al, 2018)
The xpf and ercc1 P. patens mutants are viable and show no phenotypic defect under normal conditions, in agreement with what was observed for the Arabidopsis xpf and ercc1 mutants (Hefner et al, 2003; Preuss and Britt, 2003; Dubest et al, 2004), but in contrast with the situation observed in mammalian cells, where null mutants of the ERCC1 or XPF genes are lethal (McWhir et al, 1993; Núñez et al, 2000; Hsia et al, 2003; Tian et al, 2004)
Like their Arabidopsis counterparts the xpf and ercc1 P. patens mutants are fully fertile suggesting that in plants, like in S. cerevisiae, this complex has only a minor role, if one, in meiosis which is in contrast with what is observed in Drosophila or C. elegans where the homologs of XPF1 have been shown to be involved in meiotic crossover formation (Sekelsky et al, 1995; Radford et al, 2005; Agostinho et al, 2013; O’Neil et al, 2013; Saito et al, 2013)
Summary
The XPF-ERCC1 complex is a highly conserved heterodimeric structure-specific endonuclease (SSE) composed of the XPF catalytic subunit and the ERCC1 DNA binding subunit that is involved in DNA repair and maintenance of chromosome stability (Dehé and Gaillard, 2017; Faridounnia et al, 2018). The protein sequences of the different ERCC1 homologs (ERCC1 in Drosophila melanogaster, Rad in Saccharomyces cerevisiae and Swi in Schizosaccharomyces pombe) and XPF homologs (MEI-9 in D. melanogaster, Rad in S. cerevisiae and Rad in S. pombe) are highly conserved as well as their capacity for heterodimerization, which insures stability and functionality of the complex. Consistent with the importance of heterodimerization of the two proteins for their function is that individual mutants in the ERCC1 and XPF genes exhibit similar phenotypes. Role of XPF-ERCC1 in DNA Repair in Physcomitrella (Gregg et al, 2011). It was demonstrated that XPFERCC1 participates in the Fanconi Anemia Pathway of DNA interstrand crosslinks repair (Bhagwat et al, 2009) and recently the XPF-ERCC1 complex has been shown to be involved in a subpathway of long-patch base excision repair (BER) involving 5 gap formation (Woodrick et al, 2017)
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