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

Abstract

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.A ll 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 Ex...

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…EXO1 catalyzes long-range resection of MRN-processed DNA ends in human cells (97, 98). However, EXO1 is strongly inhibited by replication protein A (RPA) binding to the resulting ssDNA (99, 100). Single-molecule imaging identified an additional, noncatalytic role for MRN in long-range resection.…”

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

“…However, the average increase in the telomeric overhang signal is only twofold, which, assuming that the telomeric overhangs are in the 35-to 600-nucleotide range , appears insufficient to explain the observed telomere truncations. However, as Exo1 is processive in vitro (Nimonkar et al 2011;Myler et al 2016), there may be a wide range in overhang lengths in cells lacking normal CST function. Indeed, in fungi and plants, impaired CST function results in extensive degradation of the 5 ′ end of telomeres (Garvik et al 1995;Martin et al 2007;Song et al 2008).…”

PHF11 promotes DSB resection, ATR signaling, and HR

“…Since previous reports noted the inhibition of EXO1 by RPA (Genschel and Modrich 2003;Myler et al 2016), we first examined the role of PHF11 in the context of this RPA-EXO1 interplay. When dsDNA with a 100-nt 3 ′ overhang (the preferred entry site for EXO1) (Cannavo et al 2013) was examined, RPA had a strong inhibitory effect on EXO1 (Fig.…”