Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice

Tomimatsu, Nozomi; Mukherjee, Bipasha; Hardebeck, Molly Catherine; Ilcheva, Mariya; Camacho, Cristel V.; Harris, Janelle L.; Porteus, Matthew H.; Llorente, Bertrand; Khanna, Kum Kum; Burma, Sandeep

doi:10.1038/ncomms4561

Cited by 161 publications

(142 citation statements)

References 46 publications

Supporting

Mentioning

139

Contrasting

Unclassified

Order By: Relevance

“…As hExo1 cannot resect past a nucleosome in vitro (66), RPA may also be required for recruiting chromatin remodelers to the DSB ahead of the resection machinery. Exo1 is also subject to a growing list of posttranslational modifications and is positively and negatively regulated by MRN, BLM, CtIP, and other components of the resection machinery (10,21,31,35,36). Our work provides a framework for future studies to determine how these interactions facilitate long-range DNA resection by Exo1 in the presence of RPA.…”

Section: Discussionmentioning

confidence: 87%

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

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: Discussionmentioning

confidence: 87%

“…When tagged at the C terminus, hExo1 retains full biochemical activity in vitro and is active in vivo. Therefore, we purified the enzyme with a C-terminal biotinylation sequence from cells overexpressing biotin ligase (15,(34)(35)(36). Nearly 100% of the hExo1 molecules were biotinylated (as measured by streptavidin band-shift; Fig.…”

Section: Resultsmentioning

confidence: 99%

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

“…This activation of ATR and Chk1 kinases also occurs as a consequence of replication, when the replication fork generates single-strand DNA (ssDNA) (Paulsen and Cimprich, 2007). Cdk2 phosphorylates the key exonucleases CtIP (also known as RBBP8) and Exo1 to enable DNA endresection and ensure that end-resection occurs only after S-phase entry (Buis et al, 2012;Tomimatsu et al, 2014;Wang et al, 2013). In addition, Cdk2-dependent, activating phosphorylations of the ATR-interacting protein ATRIP (at S224) and Chk1 itself (at S286 and S301) further restrict full activation of ATR and Chk1 to S-and G2-phase (Myers et al, 2007;Xu et al, 2012).…”

Section: S-phasementioning

confidence: 99%

The same, only different – DNA damage checkpoints and their reversal throughout the cell cycle

Shaltiël

Krenning

Bruinsma

et al. 2015

Journal of Cell Science

254

272

View full text Add to dashboard Cite

Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damageinduced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made.

show abstract

“…HR is more active in S and G 2 phases during DNA replication, since an identical sister chromatid is available as a template for repair (6,12). A major restriction point in the choice between DSB repair pathways is the competition between Ku-mediated DNA end protection and MRN complex-initiated DNA end resection (13,14). For example, initiation of 5′-3′ resection of DNA ends by the MRN complex commits cells to HR-dependent repair, and prevents repair by classical NHEJ.…”

Section: Introductionmentioning

confidence: 99%

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

Guo¹,

Magis²,

Xu³

et al. 2017

Journal of Clinical Investigation

View full text Add to dashboard Cite

Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice

Cited by 161 publications

References 46 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 same, only different – DNA damage checkpoints and their reversal throughout the cell cycle

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

Contact Info

Product

Resources

About