Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.All DNA maintenance processes require nucleases, which enzymatically cleave the phosphodiester bonds in nucleic acids. Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1–3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2, 4–7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8, 9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1, 10–12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis. The central role of Exo1 in DNA repair is highlighted by the large set of genetic interactions between Exo1 and nearly all other DNA maintenance and metabolism pathways (13).Exo1 generates long tracts of ssDNA in both MMR and DSB repair (3). This ssDNA is rapidly bound by replication protein A (RPA), a ubiquitous heterotrimeric protein that participates in all DNA transactions that generate ssDNA intermediates (14). RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15–17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/DNA2- and Exo1-dependent DNA resection pathways (18–21). Reconstitution of both the yeast and human BLM (Sgs1 in yeast)/DNA2-dependent resection reactions established that RPA stimulates DNA unwinding by BLM/Sgs1 and enforces a 5′-endonuclease polarity on DNA2 (20, 22). However, the effect of RPA on Exo1 remains unresolved. Independent studies using reconstituted yeast proteins reported that RPA could both inhibit (23) and stimulate yeast Exo1 (yExo1) (18). Similarly, human RPA has variously been reported to stimulate (19) or inhibit human Exo1 (hExo1) (4, 5, 21).In addition to RPA, human cells also encode SOSS1, a heterotrimeric ssDNA-binding complex that is essential for HR (24). SOSS1 consists of INTS3 (SOSSA), hSSB1 (SOSSB1), and C9orf80 (SOSSC) (24–26). SOSSB1 encodes a single ssDNA-binding domain that bears structural homology to Escherichia coli ssDNA-binding protein (SSB) (24). SOSS1 foci form rapidly after induction of DNA breaks, and ablation of SOSS1 severely reduces DNA resection, γH2AX foci formation, and HR at both ionizing radiation- and restriction endonuclease-induced DSBs (12, 24, 25, 27). In vitro, SOSS1 stimulates hExo1-mediated DNA resection and may help to load hExo1 at ss/dsDNA junctions (21). However, the functional relationship between SOSS1 and RPA during hExo1 resection remains unresolved.Here, we use high-throughput single-molecule DNA curtains and quantitative cell biology to reveal the interplay between human and yeast Exo1 and SSBs during DNA resection. We show that both human and yeast Exo1s are processive nucleases, but are rapidly stripped from DNA by RPA. RPA inhibition is dependent on its multiple DNA binding domains. Remarkably, SOSS1 and other SSBs with fewer than three DNA binding domains support long-range resection by hExo1. In human cells, depletion of RPA increases the rate of hExo1 recruitment to laser-induced DNA damage but reduces the extent of resection. In the presence of RPA, both human and yeast Exo1 can resect DNA using a distributive, multiple-turnover mechanism, potentially reconciling prior conflicting in vitro observations. Together, our work reveals the mechanistic basis for how RPA and SOSS1 differentially modulate hExo1 activity and highlights an additional, unexpected role for these SSBs in DNA resection. We anticipate that these findings will shed light on how Exo1 is regulated in multiple genome maintenance pathways.