PCNA promotes processive DNA end resection by Exo1

Chen, Xiaoqing; Paudyal, Sharad C.; Chin, R; You, Zhipeng

doi:10.1093/nar/gkt672

Cited by 68 publications

(89 citation statements)

References 73 publications

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“…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%

“…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%

“…In addition, Bowen et al reported that mismatch-provoked resection by scExo1 was limited in the presence of scRPA, but was strongly stimulated by scMutSα (66). During HR, hExo1 is stimulated by the protein complex Mre11/Rad50/Nbs1 (Nibrin), (MRN), BLM, and proliferating cell nuclear antigen (PCNA)-all of which physically interact with hExo1 and may help to retain the nuclease on DNA (19,21,36). Furthermore, we have shown that in the presence of RPA, both human and yeast Exo1 can rebind the same DNA site multiple times, suggesting a distributive resection mechanism (Fig.…”

Section: Discussionmentioning

confidence: 99%

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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.

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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: Discussionmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

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.

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“…Preparation of Recombinant Proteins-PCNA-His 6 (24), and the GST-pol C proteins were obtained by inducing transformed Rosetta (DE3) pLysS Escherichia coli cells with 1 mM isopropyl 1-thio-␤-Dgalactopyranoside when the culture reached OD 600 Ϸ 0.6 and culturing at 37°C for 4 h. Subsequently, the PCNA-His 6 and GST-pol C proteins were purified by using Talon affinity resin (Clontech) and glutathione agarose (Pierce), respectively, following the manufacturers' recommended procedures.…”

Section: Methodsmentioning

confidence: 99%

The Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance

Dai

You

Wang

2016

Molecular & Cellular Proteomics

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DNA polymerase (pol ) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (Ser 687 ), which is located in the highly conserved nuclear localization signal (NLS) region of human pol and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of pol with proliferating cell nuclear antigen (PCNA). Furthermore, we demonstrated that the phosphorylation of Ser 687 in pol confers cellular protection from UV irradiation and increases the efficiency in replication across a site-specifically incorporated cyclobutane pyrimidine dimer in human cells. Based on these results, we proposed a mechanistic model where Ser 687 phosphorylation functions in the reverse polymerase switching step of translesion synthesis: The phosphorylation brings negative charges to the NLS of pol , which facilitates its departure from PCNA, thereby resetting the replication fork for highly accurate and processive DNA replication. Thus, our study, together with previous findings, supported that the posttranslational modifications of NLS of pol played a dual role in polymerase switching, where Lys 682 deubiquitination promotes the recruitment of pol to PCNA immediately prior to lesion bypass and Ser 687 phosphorylation stimulates its departure from the replication fork immediately after lesion bypass. Molecular & Cellular

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“…Forty-eight hours after infection, DNA damage was introduced to cells via irradiation with 551-nm dye laser in a line pattern. A customized laser microirradiation system was previously described (Pei et al 2011;Chen et al 2013). Briefly, the system consists of an inverted microscope (Nikon), a laser ablation unit (Photonic Instruments), and microscope automation and imaging software (Metamorph, Molecular Devices).…”

Section: In Vitro Ubiquitylation and Deubiquitylation Assaysmentioning

confidence: 99%

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

et al. 2016

Self Cite

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Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA doublestrand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A-H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response.

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PCNA promotes processive DNA end resection by Exo1

Cited by 68 publications

References 73 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 Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

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