The Recombination-deficient Mutant RPA (rfa1-t11) Is Displaced Slowly from Single-stranded DNA by Rad51 Protein

Kantake, Noriko; Sugiyama, Tomohiko; Kolodner, Richard D.; Kowalczykowski, Stephen C.

doi:10.1074/jbc.m302995200

Cited by 88 publications

(93 citation statements)

References 55 publications

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“…The RPA complex containing RPA1-t11 also had high affinity binding for ssDNA (K a ϭ 140 ϫ 10 8 M Ϫ1 ; Fig. 3C; Table 2), similar to findings for Rfa1-t11 in yeast (32). These data demonstrate that forms of RPA with a deletion of either terminus or the t11 mutation (R41E, Y42F) in DBD-F are stable in vitro and have high affinity ssDNA binding activity.…”

Section: The Rpa1 Knockdown Phenotype Can Be Rescued By Exogenous Expsupporting

confidence: 78%

“…Despite this, deletion of either the N terminus or C terminus of the yeast RPA1 homolog is lethal (28). Additionally, the rfa1-t11 point mutation in the RPA1 gene in yeast leads to defects in DNA repair, recombination, and cell cycle regulation but not DNA replication (20,(31)(32)(33)35). To examine the contribution of these domains to RPA1 function in human cells, we generated a mutation in the N terminus of RPA1 (RPA1-t11; R41E and Y42F) analogous to rfa1-t11 in yeast, a deletion of the N terminus (RPA1-⌬FL; deletion of DBD-F and linker), and a deletion of the C terminus (DBD-⌬C; deletion of DBD-C) ( Fig.…”

Section: The Rpa1 Knockdown Phenotype Can Be Rescued By Exogenous Expmentioning

confidence: 99%

“…One mutation that has been extensively studied is rfa1-t11, which is functional for DNA replication and cell viability (20) but is defective in recombination and cell cycle regulation after DNA damage (13,32). The analogous mutation in human RPA1 shows near wild-type ssDNA binding affinity ( Fig.…”

Section: The Rpa1 Knockdown Phenotype Can Be Rescued By Exogenous Expmentioning

confidence: 99%

“…There is little contribution of DBD-F to ssDNA binding (32,46), so this domain must be important for other processes involving RPA. Although Philipova et al (28) demonstrated that deletion of more than the first 10 amino acids of this domain in yeast results in cell inviability, we have shown that RPA1-⌬FL in unstressed human cells appears to be indistinguishable from WT RPA1 and from RPA1-t11.…”

Section: Mutation Of Key Aromatic Residues Of Rpa1 Reveals the Importmentioning

confidence: 99%

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Cellular Functions of Human RPA1

Haring

Mason

Binz³

et al. 2008

Journal of Biological Chemistry

102

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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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Section: The Rpa1 Knockdown Phenotype Can Be Rescued By Exogenous Expsupporting

confidence: 78%

Section: The Rpa1 Knockdown Phenotype Can Be Rescued By Exogenous Expmentioning

confidence: 99%

Section: The Rpa1 Knockdown Phenotype Can Be Rescued By Exogenous Expmentioning

confidence: 99%

Section: Mutation Of Key Aromatic Residues Of Rpa1 Reveals the Importmentioning

confidence: 99%

See 2 more Smart Citations

Cellular Functions of Human RPA1

Haring

Mason

Binz³

et al. 2008

Journal of Biological Chemistry

102

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“…This ssDNA region is believed to be complexed with RPA, which provides a binding site for RFC for subsequent PCNA loading. However, our DL substrate had only a one-nucleotide ssDNA region at the 3′ end of the invading DNA strand, which is too small for RPA binding (48,49) yet required RPA for PCNA loading. Therefore, we performed a series of experiments to identify the location of RPA in PCNA loading using different DNA structures ( Fig.…”

mentioning

confidence: 99%

PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

Holzschu

Sugiyama

2013

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

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Proliferating cell nuclear antigen (PCNA) is required for DNA homologous recombination (HR), but its exact role is unclear. Here, we investigated the loading of PCNA onto a synthetic D-loop (DL) intermediate of HR and the functional interactions of PCNA with Rad51 recombinase and DNA polymerase (Pol) δ, Pol η, and Pol ζ. PCNA was loaded onto the synthetic DL as efficiently as it was loaded onto a primed DNA substrate. Efficient PCNA loading requires Replication Protein A, which is associated with the displaced ssDNA loop and provides a binding site for the clamp-loader Replication Factor C. Loaded PCNA greatly stimulates DNA synthesis by Pol δ within the DL but does not affect primer recognition by Pol δ. This suggests that the essential role of PCNA in HR is not recruitment of Pol δ to the DL but stimulation of Pol δ to displace a DNA strand during DL extension. Both Pol η and Pol ζ extended the DL more efficiently than Pol δ in the absence of PCNA, but little or no stimulation was observed in the presence of PCNA. Finally, Rad51 inhibited both the loading of PCNA onto the DL and the extension of the DL by Pol δ and Pol η. However, preloaded PCNA on the DL counteracts the Rad51-mediated inhibition of the DL extension. This suggests that the inhibition of postinvasion DNA synthesis by Rad51 occurs mostly at the step of PCNA loading.DNA repair | translesion polymerase | sliding clamp D NA double-strand breaks (DSBs) are introduced into the genome by several factors, including ionizing radiation, mutagenic chemicals, reactive oxygen species, and stalled DNA replication (1). Without appropriate repair, DSBs may lead to cell lethality or cancer (2-4). Homologous recombination (HR) is a widely conserved essential mechanism for high-fidelity repair of DSBs (5-8). HR is a highly coordinated multistep biochemical process, which is most elegantly demonstrated in the yeast Saccharomyces cerevisiae. Briefly, DSB ends are first processed by specialized exonucleases to generate 3′ overhangs (9-11), which are then coated with the ssDNA binding protein Replication Protein A (RPA). The Rad52 recombination mediator interacts with RPA and recruits Rad51 recombinase onto the ssDNA to form a helical nucleoprotein filament (12-15). This Rad51-ssDNA filament mediates strand invasion into a homologous dsDNA (16) to produce a DNA structure that is referred to as the D-loop (DL; see Fig. 6B). The 3′ end of the invading strand is used as the primer by DNA polymerases for DNA synthesis (postinvasion DNA synthesis), extending the DL (see Fig. 6F). After postinvasion DNA synthesis, repair may be completed by a break-induced replication, DSB repair, or synthesis-dependent strand-annealing pathway (5).Postinvasion DNA synthesis is crucial to high-fidelity repair of DSBs because it recovers the genetic information that might be lost during breakage events. Several DNA polymerases, including polymerase (Pol) δ, Pol η, and Pol ζ, are possibly involved in this process. Pol δ is known as a replicative polymerase that constitutes the replisome (17,...

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Replication protein A: Single‐stranded DNA's first responder

Chen

Wold²

2014

BioEssays

252

167

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Summary Replication Protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis.

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The Recombination-deficient Mutant RPA (rfa1-t11) Is Displaced Slowly from Single-stranded DNA by Rad51 Protein

Cited by 88 publications

References 55 publications

Cellular Functions of Human RPA1

Cellular Functions of Human RPA1

PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

Replication protein A: Single‐stranded DNA's first responder

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