Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

Sertic, Sarah; Pizzi, Sara; Cloney, Ross; Lehmann, Alan R.; Marini, Federica; Plevani, Paolo; Muzi-Falconi, Marco

doi:10.1073/pnas.1108547108

Cited by 69 publications

(79 citation statements)

References 30 publications

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“…Fifty-six percent of all hExo1 molecules (n = 244/435) localized to the vicinity of the 3′-ssDNA ends (Fig. 1C); the remaining nucleases were distributed at internal binding sites, consistent with hExo1's role in binding DNA nicks during MMR and NER (7,38). These data indicate that individual hExo1 molecules preferentially bind 3′-ssDNA overhangs but can also bind rare DNA nicks (we estimate approximately three to five per DNA molecule) that occur as a result of handling the 48-kb-long λ-DNA substrates.…”

Section: Resultsmentioning

confidence: 72%

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

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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...

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Section: Resultsmentioning

confidence: 72%

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

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“…Similarly, it is plausible that NER-mediated ssDNA gaps in quiescent cells may need additional enzymatic processing to activate ATR for H2AX phosphorylation. In fact, ATR-mediated checkpoint activation has been shown to require the gap enlargement of NER intermediates by Exo1, using yeast Exo1 mutant (36), Exo1-down-regulated human cell lines (37), or more clearly a defined in vitro system (38). The mechanism underlying the NER-dependent DSB formation is currently unknown and awaits further study.…”

Section: Discussionmentioning

confidence: 99%

Nucleotide Excision Repair-dependent DNA Double-strand Break Formation and ATM Signaling Activation in Mammalian Quiescent Cells

Wakasugi

Sasaki

Matsumoto

et al. 2014

Journal of Biological Chemistry

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Background:In quiescent human cells, UV induces histone H2AX phosphorylation by ATR in a nucleotide excision repair (NER)-dependent manner. Results: UV also activates ATM in response to NER-mediated DNA double-strand break (DSB). Conclusion:The NER reaction in quiescent cells potentially generates multiple types of secondary DNA damage. Significance: This work highlights the importance of our understanding of the DNA damage response in quiescent cells.

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“…However, when it fails to repair certain relatively resistant UV lesions, resection of the damaged DNA and processing of the surrounding chromatin take place. This results in checkpoint activation and engagement of alternative repair mechanisms (Giannattasio et al 2010;Sertic et al 2011). The delayed kinetics and the requirement for relatively high doses of irradiation for the localization of overexpressed, endogenous BRCA1 protein at UV lesions in G0/G1 cells suggested that recruitment in this instance was directed to those UV damage sites where NER was incomplete.…”

Section: Mir-545 Modulates the Efficiency Of Brca1-driven Hrrmentioning

confidence: 99%

“…Hence, we studied the effect of depleting G0/G1 cells of EXO1, an exonuclease that plays a role in UV-induced checkpoint activation in quiescent cells (Sertic et al 2011).…”

Section: Mir-545 Modulates the Efficiency Of Brca1-driven Hrrmentioning

confidence: 99%

Physiological modulation of endogenous BRCA1 p220 abundance suppresses DNA damage during the cell cycle

Dimitrov

Naetar

et al. 2013

Genes Dev.

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Endogenous BRCA1 p220 expression peaks in S and G2 when it is activated, and the protein participates in certain key DNA damage responses. In contrast, its expression is markedly reduced in G0/G1. While variations in transcription represent a significant part of p220 expression control, there is at least one other relevant process. We found that a microRNA, miR-545, that is expressed throughout the cell cycle down-modulates endogenous p220 mRNA and protein abundance directly in both G0/G1 and S/G2. When miR-545 function was inhibited by a specific antagomir, endogenous p220 expression increased in G0/G1, and aberrant p220-associated DNA damage responses and de novo DNA strand breaks accumulated. Analogous results were observed upon inhibition of miR-545 function in S/G2. Both sets of antagomir effects were mimicked by infecting cells with a p220 cDNA-encoding adenoviral vector. Thus, strand breaks were a product of p220 overexpression, and their prevention by miR-545 depends on its modulation of p220 expression. Breaks were also dependent on aberrant, overexpressed p220-driven recruitment of RAD51 to either spontaneously arising or mutagen-based DNA damage sites. Hence, when its level is not physiologically maintained, endogenous p220 aberrantly directs at least one DNA repair protein, RAD51, to damage sites, where their action contributes to the development of de novo DNA damage. Thus, like its loss, a surfeit of endogenous p220 function represents a threat to genome integrity.

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Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

Cited by 69 publications

References 30 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

Nucleotide Excision Repair-dependent DNA Double-strand Break Formation and ATM Signaling Activation in Mammalian Quiescent Cells

Physiological modulation of endogenous BRCA1 p220 abundance suppresses DNA damage during the cell cycle

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