Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family

Orans, Jillian; McSweeney, Elizabeth; Iyer, Ravi; Hast, Michael A.; Hellinga, Homme W.; Modrich, Paul; Beese, L.S.

doi:10.1016/j.cell.2011.03.005

Cited by 140 publications

(302 citation statements)

References 69 publications

Supporting

Mentioning

285

Contrasting

Order By: Relevance

“…As expected, nuclease-dead Exo1(D78A/D173A) bound at the 3′-ssDNA ends but did not move on DNA (mean velocity = 0.1 ± 0.5 bp/s; processivity = 0.01 ± 0.3 kb, n = 19; Fig. 1E) (32). We also did not observe any resection when EDTA was substituted for divalent metal ions (Fig.…”

Section: Resultssupporting

confidence: 48%

“…Stationary hExo1 may stem from protein inactivation during overexpression and purification or may be an intrinsic property of the enzyme. In support of the second model, a recent X-ray crystallographic study of hExo1 suggested that the largely unstructured C terminus, which is present in our full-length protein, harbors an auto-inhibitory domain (32). This domain interacts with hMSH2 and hMLH1 (40,41) and is critical for hMutSα stimulation of hExo1 nuclease activity (5,32,42).…”

Section: Resultsmentioning

confidence: 73%

“…Nanofabricated chrome barriers were used to organize thousands of DNA molecules for high-throughput data collection and analysis (28)(29)(30). As hExo1 has been reported to preferentially load on 3′-ssDNA ends (18,(31)(32)(33), we prepared a λ-phage DNA that contained a 72-nt 3′-polyT overhang. The surface-immobilized DNA was extended for fluorescent imaging via the application of mild buffer flow from a computer-controlled syringe pump (28).…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

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

Myler

Gallardo

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

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...

show abstract

Section: Resultssupporting

confidence: 48%

Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Myler

Gallardo

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

show abstract

“…Analysis of the structures of FEN1 (Tsutakawa et al 2011) and EXO1 (Orans et al 2011), which were solved at similar times, suggest a unified mechanism for this type of nuclease involving DNA bending and two nucleotide 'fraying' of one strand (Orans et al 2011).…”

Section: Fen1mentioning

confidence: 92%

The role of DNA exonucleases in protecting genome stability and their impact on ageing

Mason

Cox

2011

AGE

View full text Add to dashboard Cite

Exonucleases are key enzymes involved in many aspects of cellular metabolism and maintenance and are essential to genome stability, acting to cleave DNA from free ends. Exonucleases can act as proofreaders during DNA polymerisation in DNA replication, to remove unusual DNA structures that arise from problems with DNA replication fork progression, and they can be directly involved in repairing damaged DNA. Several exonucleases have been recently discovered, with potentially critical roles in genome stability and ageing. Here we discuss how both intrinsic and extrinsic exonuclease activities contribute to the fidelity of DNA polymerases in DNA replication. The action of exonucleases in processing DNA intermediates during normal and aberrant DNA replication is then assessed, as is the importance of exonucleases in repair of double-strand breaks and interstrand crosslinks. Finally we examine how exonucleases are involved in maintenance of mitochondrial genome stability. Throughout the review, we assess how nuclease mutation or loss predisposes to a range of clinical diseases and particularly ageing.

show abstract

“…The Mlh1-Pms1/ Pms2 complex has an endonuclease activity suggested to play a role in the initiation of the excision step of MMR (24,25). Downstream of mismatch recognition is a mispair excision step that can be catalyzed by Exonuclease 1 (Exo1) (26)(27)(28); however, defects in both S. cerevisiae and mouse Exo1 result in only a partial MMR deficiency, suggesting the existence of additional excision mechanisms (26,27,29). DNA polymerase δ, the singlestrand DNA binding protein replication protein A (RPA), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC) are also required for MMR at different steps, including activation of Mlh1-Pms1/Pms2, stimulation of Exo1, potentially in Exo1-independent mispair excision, and in the gap-filling resynthesis steps of MMR (3,16,17,24,27,(30)(31)(32)(33)(34)(35)(36).…”

mentioning

confidence: 99%

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

Bowen¹,

Smith²,

Srivatsan³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispaircontaining DNA substrates. A mixture of MutS homolog 2 (Msh2)-MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2-MutS homolog 3 mispair recognition protein could substitute for the Msh2-Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1-postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA replication fidelity | genome instability | mutator phenotype | cancer | mutagenesis

show abstract

Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family

Cited by 140 publications

References 69 publications

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

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

The role of DNA exonucleases in protecting genome stability and their impact on ageing

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

Contact Info

Product

Resources

About