A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

Glanzer, Jason G.; Carnes, Katie A.; Soto, Patricia; Liu, Shengqin; Parkhurst, Lawrence J.; Oakley, Gregory G.

doi:10.1093/nar/gks1291

Cited by 29 publications

(35 citation statements)

References 62 publications

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“…Biochemical and cell biology studies with truncated proteins have established that DBD-A and DBD-B have the strongest ssDNA-binding affinities and are essential for proper RPA function in DNA replication and repair (16,47,48). RPA1 also encodes an N-terminal DBD-F, which is connected to the DBD-A/B via a long polypeptide linker, harbors a weak DNA binding activity, and physically interacts with DNA replication and repair proteins (45,49,50). To determine the key RPA domains that are required for hExo1 displacement, we purified and assayed a series of truncated hRPA variants (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

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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: 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

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“…The HAMNO structure used for docking was created using Open Babel. The model of DBD-F chosen for docking was a modified version of the original crystal structure (PDB:2B29) optimized for ligand binding (22, 23). The entire protein structure was used for modeling under default conditions, resulting in five simulating dockings of high predictive affinity.…”

Section: Methodsmentioning

confidence: 99%

“…DBD-F fused to maltose binding protein was generated and purified as described (22). Quality of both proteins were assessed by SDS-PAGE, followed by coomassie staining (22).…”

Section: Methodsmentioning

confidence: 99%

RPA Inhibition Increases Replication Stress and Suppresses Tumor Growth

et al. 2014

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The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions which rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress.

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“…MLN4924 inhibits the Nedd8 activating enzyme (NAE) and has been shown to inhibit the neddylation of CtIP which leads to an increase of the extent of DNA end resection at strand breaks thereby promoting HDR 27 by leaving ssDNA stabilized by RPA that can undergo recombination. NSC15520 prevents the association of RPA with p53 and RAD9 28,29 , possibly increasing the abundance of RPA available which could favor HDR.…”

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confidence: 99%

Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Riesenberg

Maričić

2017

Preprint

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A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks (DSBs) which are then repaired by homology-directed repair (HDR) using synthetic or cloned DNA donor molecules carrying desired mutations. However, another pathway, the non-homologous end joining (NHEJ) pathway competes with HDR for repairing DNA breaks in cells. To increase the frequency of precise genome editing in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) we have tested the capacity of a number of small molecules to enhance HDR or inhibit NHEJ. We identify molecules that increase the frequency of precise genome editing including some that have additive effects when applied together. Using a mixture of such molecules, the 'CRISPY' mix, we achieve 2.8-to 6.7-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes, to our knowledge the highest editing efficiency in hiPSCs described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.9-to 4.0-fold, allowing almost 20% of chromosomes to be edited. Main textThe bacterial nuclease CRISPR/Cas9 is now frequently used to accurately cut chromosomal DNA sequences in eukaryotic cells. The resulting DNA breaks are repaired by two competing pathways: NHEJ and HDR (Fig. 1). In NHEJ, the first proteins to bind the cut DNA ends are Ku70/Ku80, followed by DNA protein kinase catalytic subunit (DNA-PKcs) Since NHEJ of Cas9-induced DSBs is error prone and frequently introduces short insertions and deletions (indels) at the cut site, it is useful for knocking out a targeted gene. In contrast, HDR allows precise repair of a DSB by using a homologous donor DNA. If the donor DNA provided in the experiment carries mutations, these will be introduced into the genome (precise genome editing). Repair with homologous ssDNA or dsDNA has been suggested to engage different pathways 5 . We will refer to Targeted Nucleotide Substitutions using ssDNA donors as 'TNS' and targeted insertion of cassettes . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/136374 doi: bioRxiv preprint first posted online May. 10, 2017; 3 using dsDNA donors as knock-ins, respectively. In order to introduce a DSB Cas9 requires the nucleotide sequence NGG (a "PAM" site) in the target DNA. Targeting of Cas9 is further determined by a guide RNA (gRNA) complementary to 20 nucleotides adjacent to the PAM site. However, the Cas9 may also cut the genome at sites that carry sequence similarity to the gRNA 6 . One strategy to reduce such off-target cuts is to use a mutated Cas9 that introduces single-stranded nicks instead of DSBs (Cas9n) 7 . Using two gRNAs to introduce two nicks on opposite DNA strands in close proximity to each other will result in a staggered D...

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A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

Cited by 29 publications

References 62 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

RPA Inhibition Increases Replication Stress and Suppresses Tumor Growth

Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

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