The 32- and 14-Kilodalton Subunits of Replication Protein A Are Responsible for Species-Specific Interactions with Single-Stranded DNA

Sibenaller, Zita A.; Sorensen, Brenda R.; Wold, Marc S.

doi:10.1021/bi981110+

Cited by 72 publications

(80 citation statements)

References 64 publications

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“…Thus, at equilibrium, the scRPA protein does not appear to bind with very high cooperativity under our solution conditions in either binding mode. Our ITC and sedimentation equilibrium studies also support this conclusion and thus agree with the studies of Sibenaller et al (48).…”

Section: Discussionsupporting

confidence: 94%

“…The scRPA hetero-trimer was expressed and purified as described (48) with the following modification. ScRPA was eluted from the anion exchange column (Mono Q 5/50 GL, Pharmacia) using a 10 mL linear KCl gradient from 100 to 400 mM KCl.…”

Section: Scrpa Protein and Nucleic Acidsmentioning

confidence: 99%

“…The purified protein was dialyzed into buffer T plus 0.1 M NaCl, 0.2 mM 2 mercaptoethanol, and 30%(v/v) glycerol, and stored at −20°C. The scRPA concentration was determined spectrophotometrically in buffer T (0.02 M NaCl) by using an extinction coefficient of 9.85 × 10 4 M −1 cm −1 (48).…”

Section: Scrpa Protein and Nucleic Acidsmentioning

confidence: 99%

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Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

2006

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We have examined the single stranded DNA binding properties of the S. cerevisiae Replication Protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry and sedimentation equilibrium in order to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants and binding enthalpy of scRPA-((dT) L ) complexes as a function of oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is stable heterotrimer over the range of [NaCl] examined (0.02 M to 1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n=18−20 nucleotides at low [NaCl] to n=26 −28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0°C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT) L complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in occluded site size. Measurements of the ΔH obs for scRPA binding to (dT) L at 1.5 M NaCl, yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 ± 1 nucleotides), stabilized at low [NaCl], in which only three of its OB-folds are used, and a higher site size mode (n = 27 ± 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found either under any conditions examined. Thus, scRPA shows some similar behavior to the E. coli SSB homo-tetramer, which also shows binding mode transitions, but some significant differences also exist.Single stranded (ss) DNA binding (SSB) proteins exist in nearly all organisms and play central roles in all aspects of DNA metabolism, including DNA replication, repair, and recombination. However, the structural forms of SSB proteins differ considerably among different organisms. For example, the bacteriophage T4 gene 32 protein, the first such SSB protein identified (1, 2) is a monomer, whereas the E. coli SSB protein, is a homotetramer (3,4), as are most SSB proteins from eubacteria. In contrast, the eukaryotic SSB protein, commonly called Replication Protein A (RPA) is a hetero-trimer (5,6).In all eukaryotes, RPA consists of three highly conserved subunits of approximately 70, 32, and 14 kDa, named according to their respective molecular weights, and all three subunits are required for function in vivo (5,6). However, RPA homologues display distinct species specificity, such that scRPA from yeast cannot substitute for human RPA (hsRPA) Figure showing the results of agarose gel electrophoresis experiments at low (20 mM) and high (1.5 M) NaCl concentrations for scRPA-ssDNA complexes formed at different protein to DNA ratios indicating low or no cooperativity upon formation of these complexes. This material is availa...

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Section: Discussionsupporting

confidence: 94%

Section: Scrpa Protein and Nucleic Acidsmentioning

confidence: 99%

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Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

2006

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“…Yeast Polδ variants and replication protein A (RPA) were purified as described previously (30,85). Yeast proliferating cell nuclear antigen (PCNA) was purified using DEAE-Sepharose, S-Sepharose, Mono-Q, and hydroxyapatite column chromatography using the methods described by Ayyagari et al (86) and Fien and Stillman (87).…”

Section: Methodsmentioning

confidence: 99%

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Mertz

Sharma²,

Chabes

et al. 2015

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

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Defects in DNA polymerases δ (Polδ) and e (Pole) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.DNA polymerase δ | colon cancer | dNTP pools | mutagenesis | DNA replication fidelity W hen functioning properly, DNA replication is phenomenally accurate, making ∼10 −10 mutations per base pair during each replication cycle (1). In respect to human biology, it means that, on average, less than one mutation occurs each time the human genome is replicated. This amazing exactitude is contingent upon the serial action of DNA polymerase selectivity, exonucleolytic proofreading, and DNA mismatch repair (MMR). MMR defects increase spontaneous mutagenesis in numerous model systems (2) and give rise to cancer in mice (3). In humans, inherited mutations in MMR genes predispose to colorectal cancer (CRC) in Lynch syndrome (4, 5). Additionally, MMR genes are inactivated via hypermethylation in ∼15% of sporadic CRC, endometrial cancer (EC), and gastric cancer (6).Like defects in MMR, mutations that decrease the base selectivity or proofreading activity of replicative DNA polymerases elevate spontaneous mutagenesis in eukaryotic cells (7-14) and cancer incidence in mice (15)(16)(17)(18). Germline mutations affecting the exonuclease domains of DNA polymerases δ (Polδ) and e (Pole) cause hereditary CRC (19), and somatic changes in the exonuclease domain of Pole were found in sporad...

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“…S. cerevisiae Pif1 was cloned into the pET-28b bacterial expression vector (Novagen/EMD Biosciences), expressed in the E. coli Rosetta strain (Novagen/ EMD Biosciences), and purified as previously described (33). S. cerevisiae RPA was overexpressed and purified from E. coli as previously described (34). S. cerevisiae Dna2 was overexpressed and purified from baculovirus High Five cells as previously described (23).…”

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confidence: 99%

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

Pike

Burgers

Campbell

et al. 2009

Journal of Biological Chemistry

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We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase ␦ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase ␦ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5Ј to 3Ј direction. A leading strand is synthesized by DNA polymerase ⑀ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase ␦ (pol ␦) 3 in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ϳ150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase ␣/primase (pol ␣) initiates each fragment by synthesizing an RNA/DNA primer consisting of ϳ1-nt of RNA and ϳ10 -20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol ␦ then complexes with PCNA and extends the primer. When pol ␦ reaches the 5Ј-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol ␦ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7-9). FEN1 binds at the 5Ј-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol ␦ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol ␦ and FEN1 also tends to keep flaps...

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The 32- and 14-Kilodalton Subunits of Replication Protein A Are Responsible for Species-Specific Interactions with Single-Stranded DNA

Cited by 72 publications

References 64 publications

Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

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