Replication Protein A Interactions with DNA:  Differential Binding of the Core Domains and Analysis of the DNA Interaction Surface

Wyka, Iwona M; Dhar, Kajari; Binz, Sara K.; Wold, Marc S.

doi:10.1021/bi034930h

Cited by 75 publications

(150 citation statements)

References 38 publications

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“…Although both human and yeast RPA can displace hExo1, hRPA can do so twice as rapidly as the yeast protein (Table S1). Both RPA complexes have subnanomolar affinity for ssDNA, suggesting that hRPA may displace hExo1 by direct competition for the ssDNA and also via species-specific interactions (46,47). These results are surprising, as a physical interaction between the human or yeast RPA and Exo1 has not been reported (15,21).…”

Section: Resultsmentioning

confidence: 98%

“…2F, Upper) (14, 47). 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).…”

Section: Resultsmentioning

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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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: 98%

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.

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104

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“…The integrity of the RPA70-coding sequence was verified by DNA sequencing. RPA70 mutations (33) were introduced into the BRC-RPA vectors by swapping an MfeI͞ MscI restriction fragment containing the mutations. The RPA70 R234A͞R263A mutant (33) also carries a third mutation (N239K), which is inferred to be a silent mutation for DNA binding (M. Wold, personal communication).…”

Section: Methodsmentioning

confidence: 99%

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

Saeki

Siaud

Christ

et al. 2006

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

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The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.double-strand break ͉ mammalian cells ͉ Rad51 ͉ homologous recombination ͉ BRC repeat

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“…Although each OB-fold is structurally similar, the majority of the ssDNA binding occurs through two OB-folds (DBD-A and DBD-B) centrally located in RPA1, referred to as the ssDNA-binding core (17, 18). The ssDNA-binding core is both necessary and sufficient for high affinity DNA binding (17)(18)(19). In addition to the ssDNA-binding core, RPA1 contains an OB-fold at each terminus.…”

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confidence: 99%

“…RPA1 contains four OB-folds, and RPA2 and RPA3 have one OB-fold each. Although each OB-fold is structurally similar, the majority of the ssDNA binding occurs through two OB-folds (DBD-A and DBD-B) centrally located in RPA1, referred to as the ssDNA-binding core (17,18). The ssDNA-binding core is both necessary and sufficient for high affinity DNA binding (17)(18)(19).…”

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confidence: 99%

Cellular Functions of Human RPA1

Haring

Mason

Binz³

et al. 2008

Journal of Biological Chemistry

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100

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In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.Cell survival and proliferation depend on the efficient maintenance of genetic information. Human cells must accurately replicate billions of base pairs of DNA and identify and repair a wide variety of DNA lesions. One of the proteins required for the maintenance of genomic integrity is replication protein A (RPA). 3 RPA is a heterotrimeric single strand DNA (ssDNA)-binding protein, composed of 70-, 32-, and 14-kDa subunits, commonly referred to as RPA1, RPA2, and RPA3, respectively (1-3). Homologs of the three RPA subunits have been found in all eukaryotes examined (1, 4). The major biochemical activity of RPA is ssDNA binding; RPA can bind ssDNA with subnanomolar affinity (5). The ability of RPA to bind ssDNA is not sequence-specific; however, RPA has a higher affinity for pyrimidine-rich and telomeric ssDNA (6 -8).Replication protein A was originally isolated as a factor essential for in vitro DNA replication of simian virus 40 (SV40) (9 -11), and it has since been shown to be essential for a number of other DNA metabolic processes, including chromosomal DNA replication, repair, and recombination (1-3). RPA also has a role in maintenance of telomeres (7, 12) and in regulation of the cell cycle (13,14). The common feature of all of these processes is that each has ssDNA intermediates that must be recognized and processed appropriately. RPA interacts with ssDNA in the cell and with a number of proteins involved in these processes (3,15).Each of the RPA subunits has at least one domain containing an oligonucleotide/oligosaccharide-binding (OB) fold. This fold is found in many ssDNA-and sugar-binding proteins (16). The OB-folds in RPA are commonly referred to as DNA-binding domains (DBDs). RPA1 contains four OB-folds, and RPA2 and RPA3 have one OB-fold each. Although each OB-fold is structurally similar, the majority of the ssDNA binding occurs through two OB-fo...

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Replication Protein A Interactions with DNA: Differential Binding of the Core Domains and Analysis of the DNA Interaction Surface

Cited by 75 publications

References 38 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

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

Cellular Functions of Human RPA1

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