Mechanistic analysis of Xenopus EXO1's function in 5'-strand resection at DNA double-strand breaks

Abstract

The processing of DNA double-strand breaks (DSBs) into 3′ single-stranded tails is the first step of homology-dependent DSB repair. A key player in this process is the highly conserved eukaryotic exonuclease 1 (EXO1), yet its precise mechanism of action has not been rigorously determined. To address this issue, we reconstituted 5′-strand resection in cytosol derived from unfertilized interphase eggs of the frog Xenopus laevis. Xenopus EXO1 (xEXO1) was found to display strong 5′→3′ dsDNA exonuclease activity but no significant ssDNA exonuclease activity. Depletion of xEXO1 caused significant inhibition of 5′ strand resection. Co-depletion of xEXO1 and Xenopus DNA2 (xDNA2) showed that these two nucleases act in parallel pathways and by distinct mechanisms. While xDNA2 acts on ssDNA unwound mainly by the Xenopus Werner syndrome protein (xWRN), xEXO1 acts directly on dsDNA. Furthermore, xEXO1 and xWRN are required for both the initiation stage and the extension stage of resection. These results reveal important novel information on the mechanism of 5′-strand resection in eukaryotes.

14-3-3 Proteins Restrain the Exo1 Nuclease to Prevent Overresection

“…As shown previously, DNA end resection occurs on this dsDNA fragment in Xenopus NPE in the 5Ј-to-3Ј direction under proper control and can be visualized on agarose gels (33,45). As in cells, bulk DNA end resection in Xenopus NPE is carried out by the Exo1 (xExo1) and Dna2 (xDna2) nucleases acting in two parallel pathways (16,18,46,47). Correspondingly, immunodepletion of either xExo1 or xDna2 from NPE partially abolishes DNA end resection (33,47).…”

“…As in cells, bulk DNA end resection in Xenopus NPE is carried out by the Exo1 (xExo1) and Dna2 (xDna2) nucleases acting in two parallel pathways (16,18,46,47). Correspondingly, immunodepletion of either xExo1 or xDna2 from NPE partially abolishes DNA end resection (33,47).…”

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

“…Sgs1 has two human homologs, BLM and WRN (mutated in Bloom's and Werner's syndromes, respectively). BLM has been shown to be the primary resection protein in mammalian cells (39,40), although Xenopus WRN-DNA2 can also resect linear DNA substrates in vitro (41). Both BLM and WRN have P. falciparum homologs (PF3D7_0918600 and PF3D7_1429900, respectively), though the P. falciparum WRN homolog lacks the 5=-to-3= exonuclease domain that is characteristic of WRN proteins in other eukaryotes.…”

Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

“…According to this model, Mre11 [5], in cooperation with CtIP (Sae2 in yeast) [6, 7], carries out initial limited resection at the sites of DSBs. The minimally-resected DNA then serves as a template for extensive resection by two alternate “downstream” pathways involving: 1) the 5’-3’ exonuclease Exo1 alone or 2) the helicases BLM or WRN (Sgs1 in yeast) in conjunction with either Exo1 or the endonuclease Dna2 [8–12]. This model has been further refined by recent biochemical studies using purified yeast or human components showing that Mre11/CtIP also stimulate resection by promoting the association of Exo1 or BLM with DNA ends [13–15].…”