FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1982181829> ?p ?o ?g. }

• W1982181829 endingPage "21768" @default.
• W1982181829 startingPage "21758" @default.
• W1982181829 abstract "Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability. Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability. Replication protein A (RPA) 2The abbreviations used are: RPA, replication protein A; nt, nucleotide(s); ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; DBD, DNA binding domain; NC, nucleocapsid protein. 2The abbreviations used are: RPA, replication protein A; nt, nucleotide(s); ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; DBD, DNA binding domain; NC, nucleocapsid protein. is a single-stranded DNA-binding protein that participates in multiple processes in eukaryotes, including DNA replication, DNA repair, and recombination (1Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1176) Google Scholar). RPA was first identified as a protein required for simian virus 40 (SV40) replication in vitro (2Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (295) Google Scholar, 3Wobbe C.R. Weissbach L. Borowiec J.A. Dean F.B. Murakami Y. Bullock P. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1834-1838Crossref PubMed Scopus (258) Google Scholar, 4Wold M.S. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar). It is known to bind and protect single-stranded DNA (ssDNA) from nucleases, assist other proteins in the unwinding of double-stranded DNA (dsDNA), and prevent the formation of stable hairpins during DNA processing (5Brosh Jr., R.M. Li J. L Kenny M. K Karow J. K Cooper M. P Kureekattil R. P Hickson I. D Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 6Brosh Jr., R.M. Orren D. K Nehlin J. O Ravn P. H Kenny M. K Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 7Yuzhakov A. Kelman Z. Hurwitz J. O'Donnell M. EMBO J. 1999; 18: 6189-6199Crossref PubMed Scopus (167) Google Scholar). Human RPA is a stable heterotrimeric complex consisting of subunits of 70, 32, and 14 kDa. The core of this trimeric protein readily binds to ssDNA sequences with a defined 5′-3′ polarity (8de Laat W.L. Appeldoorn E. Sugasawa K. Weterings E. Jaspers N.G. Hoeijmakers J.H. Genes Dev. 1998; 12: 2598-2609Crossref PubMed Scopus (264) Google Scholar, 9Iftode C. Borowiec J.A. Biochemistry. 2000; 39: 11970-11981Crossref PubMed Scopus (57) Google Scholar). RPA contains six DNA-binding domains (DBD) distributed among the three subunits. The RPA70 subunit has the highest affinity for ssDNA with four DBDs, whereas the two smaller subunits have one DBD each (10Daughdrill G.W. Ackerman J. Isern N.G. Botuyan M.V. Arrowsmith C. Wold M.S. Lowry D.F. Nucleic Acids Res. 2001; 29: 3270-3276Crossref PubMed Scopus (41) Google Scholar, 11Gao H. Cervantes R.B. Mandell E.K. Otero J.H. Lundblad V. Nat. Struct. Mol. Biol. 2007; 14: 208-214Crossref PubMed Scopus (229) Google Scholar). Additionally, the N-terminal domain of RPA70 (DBD F) has been shown to be important for binding and melting double-stranded DNA, and for helix destabilization of short DNA strands (12Lao Y. Lee C.G. Wold M.S. Biochemistry. 1999; 38: 3974-3984Crossref PubMed Scopus (96) Google Scholar). Current models of RPA binding suggest that the domains actually used for binding are contingent on the length of the ssDNA to which the RPA is bound (13Bastin-Shanower S.A. Brill S.J. J. Biol. Chem. 2001; 276: 36446-36453Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Bochkareva E. Belegu V. Korolev S. Bochkarev A. EMBO J. 2001; 20: 612-618Crossref PubMed Scopus (137) Google Scholar, 15Bochkareva E. Korolev S. Lees-Miller S.P. Bochkarev A. EMBO J. 2002; 21: 1855-1863Crossref PubMed Scopus (231) Google Scholar). Structural studies have shown that two DBDs of RPA70 (DBD A and DBD B) can interact with an 8-nt segment of ssDNA and that longer DNAs interact with additional domains. The DBD located on RPA32 (DBD D) is thought to bind to the 3′-end of the ssDNA only in the highest affinity binding mode when all four domains are utilized, although a mutation in the D site does not affect RPA binding or function in vivo (15Bochkareva E. Korolev S. Lees-Miller S.P. Bochkarev A. EMBO J. 2002; 21: 1855-1863Crossref PubMed Scopus (231) Google Scholar, 16Fanning E. Klimovich V. Nager A.R. Nucleic Acids Res. 2006; 34: 4126-4137Crossref PubMed Scopus (400) Google Scholar, 17Pestryakov P.E. Khlimankov D.Y. Bochkareva E. Bochkarev A. Lavrik O.I. Nucleic Acids Res. 2004; 32: 1894-1903Crossref PubMed Scopus (35) Google Scholar). Consequently, the role of DBD D in binding is unclear. DBD D has also been shown to bind the 3′-hydroxyl of a partial duplex (such as a primer-template junction), whereas the RPA70C domain is bound to the 5′ ssDNA overhang of the other DNA strand in the duplex. Interactions between RPA and helicases promote strand unwinding (5Brosh Jr., R.M. Li J. L Kenny M. K Karow J. K Cooper M. P Kureekattil R. P Hickson I. D Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 18Machwe A. Lozada E.M. Xiao L. Orren D.K. BMC Mol. Biol. 2006; 7: 1Crossref PubMed Scopus (40) Google Scholar, 19Garcia P.L. Liu Y. Jiricny J. West S.C. Janscak P. EMBO J. 2004; 23: 2882-2891Crossref PubMed Scopus (169) Google Scholar, 20Opresko P.L. Laine J.P. Brosh Jr., R.M. Seidman M. M Bohr V.A. J. Biol. Chem. 2001; 276: 44677-44687Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Through various protein interactions, RPA may be directly placed on ssDNA after it emerges from a helicase complex to prevent the separated strands from reannealing or forming any secondary structure. The strand unwinding activities of yeast Srs2 and human RECQ1 helicases both benefit from the addition of RPA (21Cui S. Arosio D. Doherty K.M. Brosh Jr., R.M. Falaschi A. Vindigni A. Nucleic Acids Res. 2004; 32: 2158-2170Crossref PubMed Scopus (94) Google Scholar, 22Van Komen S. Reddy M.S. Krejci L. Klein H. Sung P. J. Biol. Chem. 2003; 278: 44331-44337Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Also, RPA has been shown to stimulate the unwinding activity of the Werner and Bloom syndrome helicases on long duplexes by directly interacting with the proteins (5Brosh Jr., R.M. Li J. L Kenny M. K Karow J. K Cooper M. P Kureekattil R. P Hickson I. D Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 6Brosh Jr., R.M. Orren D. K Nehlin J. O Ravn P. H Kenny M. K Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 23Doherty K.M. Sommers J.A. Gray M.D. Lee J.W. Von Kobbe C. Thoma N.H. Kureekattil R.P. Kenny M.K. Brosh Jr., R.M. J. Biol. Chem. 2005; 280: 29494-29505Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 24Shen J.C. Lao Y. Kamath-Loeb A. Wold M.S. Loeb L.A. Mech. Ageing Dev. 2003; 124: 921-930Crossref PubMed Scopus (50) Google Scholar). Yeast RPA has also been shown to stimulate Pif1 helicase activity and aid in unwinding of both DNA-DNA substrates and DNA-RNA hybrids (25Boule J.B. Zakian V.A. Nucleic Acids Res. 2007; 35: 5809-5818Crossref PubMed Scopus (129) Google Scholar). The analogous single-stranded DNA-binding protein from Escherichia coli has been shown to remove secondary structure from ssDNA at high magnesium concentrations, allowing RecA to form presynaptic complexes, which ultimately facilitate strand annealing and exchange (26Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Crossref PubMed Scopus (199) Google Scholar, 27Muniyappa K. Shaner S.L. Tsang S.S. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2757-2761Crossref PubMed Scopus (108) Google Scholar). Similarly, two studies showed that yeast RPA can stimulate RAD52-catalyzed annealing of ssDNA (28Sugiyama T. Zaitseva E.M. Kowalczykowski S.C. J. Biol. Chem. 1997; 272: 7940-7945Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 29Sugiyama T. New J.H. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6049-6054Crossref PubMed Scopus (259) Google Scholar). In the first, RPA was found to reduce ssDNA with secondary structure during presynaptic complex formation, inhibiting nonproductive binding of RAD52 to those regions and thus streamlining its activity (28Sugiyama T. Zaitseva E.M. Kowalczykowski S.C. J. Biol. Chem. 1997; 272: 7940-7945Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In the second, the addition of RPA stimulated RAD52-catalyzed annealing of long plasmid (2961 nt) DNA. The authors suggested that the role of RPA was to reduce secondary structure of the DNA so that it would be susceptible to the annealing and ultimately exchange activities of RAD52 (29Sugiyama T. New J.H. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6049-6054Crossref PubMed Scopus (259) Google Scholar). In DNA replication, Okazaki fragments are processed by a pathway in which the 5′-end region of each fragment is displaced into a single-stranded flap for nucleolytic removal of the RNA primer (30Kao H.I. Bambara R.A. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 433-452Crossref PubMed Scopus (77) Google Scholar, 31Rossi M.L. Purohit V. Brandt P.D. Bambara R.A. Chem. Rev. 2006; 106: 453-473Crossref PubMed Scopus (54) Google Scholar). It has been hypothesized that some flaps become long enough to be coated by RPA (30Kao H.I. Bambara R.A. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 433-452Crossref PubMed Scopus (77) Google Scholar, 32Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar, 33MacNeill S.A. Curr. Biol. 2001; 11: R842-R844Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). When we tested flap substrates with RPA-coated flaps, we were surprised to find that the RPA promoted annealing of the flap to other single-stranded template DNA (34Bartos J.D. Wang W. Pike J.E. Bambara R.A. J. Biol. Chem. 2006; 281: 32227-32239Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This result suggested that RPA alone can support rather than suppress recombination. The current study is focused on determining the conditions under which RPA can promote strand annealing. Materials—All oligonucleotides were obtained commercially from Integrated DNA Technologies (Coralville, IA). Radionucleotide [γ-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences, and the T4 polynucleotide kinase (labeling grade) was purchased from Roche Applied Science. All other reagents were of the best available commercial grade. Enzyme Expression and Purification—Recombinant human RPA was purified as described previously (35Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). Oligonucleotide Substrates—Oligomer sequences are listed in Table 1. Sequences with the secondary structure required to inhibit strand annealing were designed with the help of the mfold software (version 3.2) by Zuker and Turner, available on the World Wide Web (36Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10234) Google Scholar). Using standard procedures, substrates T1 and T2 were radiolabeled at the 5′-end with [γ-32P]ATP and T4 polynucleotide kinase. The radiolabeled strands were purified on 15% denaturing polyacrylamide gels containing 7 m urea.TABLE 1Oligonucleotide sequences (5′-3′)OligonucleotideSequenceRadiolabeled templatesT1 57-merTCG AGA CCT CTG TTT CCA AGT AAA ACG ACG GCC AGT GTG CGT AGC GTA CAA TAC GACT2 57-merTGG AAA AAA AAA AAA AAG TGA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA GGA AGGComplementary primersC1 57-merGTC GTA TTG TAC GCT ACG CAC ACT GGC CGT CGT TTT ACT TGG AAA CAG AGG TCT CGAC2 47-merACG CTA CGC ACA CTG GCC GTC GTT TTA CTT GGA AAC AGA GGT CTC GAC3 37-merCAC TGG CCG TCG TTT TAC TTG GAA ACA GAG GTC TCG AC4 20-merCTT GGA AAC AGA GGT CTC GAC5 17-merCAC TGG CCG TCG TTT TACC6 57-merCCT TCC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCA CTT TTT TTT TTT TTT CCAFlapsF1 63-merCAC TGG CCG TCG TTT TAC GGT CGT GAC TGG GAA AAC CAC CCG TCC ACC CGA CGC CAC CTC CTG Open table in a new tab Secondary Structures—All oligomers were analyzed using the mfold software (36Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10234) Google Scholar). The folding free energy change (ΔG) for each oligomer was calculated based on parameters set forth in previous work (37Peyret N. Prediction of Nucleic Acid Hybridization: Parameters and Algorithms, Ph.D. thesis. Wayne State University, Detroit, MI2000Google Scholar, 38SantaLucia Jr., J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1460-1465Crossref PubMed Scopus (2275) Google Scholar) at salt concentrations of 40 mm KCl and 4 mm MgCl2 and an ambient temperature of 37 °C. A positive free energy change predicts that the oligomer should not fold into consistent structures beyond random coils. Such oligomers are described here as having no secondary structure. Only the hydrogen-bonding configuration of the lowest free energy structure was considered for each oligomer, although in many cases there were other predicted structures with higher free energies. Enzyme Assays—Reactions were performed in buffer containing 30 mm HEPES (pH 7.5), 5% glycerol, 40 mm KCl, 0.1 mg/ml bovine serum albumin, and 4 mm MgCl2. Enzyme stocks were diluted in 30 mm HEPES (pH 7.5), 5% glycerol, 40 mm KCl, and 0.1 mg/ml bovine serum albumin. Each reaction contained 5 fmol of radiolabeled template (T1 or T2) and 20 fmol (C1, C2, C3, C4, C5 or F1) or 10 fmol (C6) of unlabeled complementary strand in a 20-μl reaction mixture with varying amounts of the enzymes as indicated in the figure legends. Reactions were initiated by mixing the labeled template and unlabeled complementary strand followed immediately by the addition of the enzyme. All assays were incubated at 37 °C for the time specified in the figure legend. Reactions were stopped by the addition of 0.25 volumes of helicase dyes (30% glycerol, 50 mm EDTA, 0.9% SDS, 0.25% bromphenol blue, and 0.25% xylene cyanole) and immediately run on a native 8% polyacrylamide gel at 25 watts for 3 h. After vacuum drying, each gel was visualized and quantitated using a GE Healthcare PhosphorImager and analyzed using ImageQuant version 5.0 software from Molecular Dynamics. In all studies, the quantitated amounts of substrates and products were utilized to calculate the percentage of product formation from the product/(product + substrate) ratio. This method allows for the correction of any loading errors among lanes. The graphed data points were calculated by quantifying the pixel density of each band and subtracting the measurable background pixel density. The percentage of strand-annealing activity was calculated as the amount of labeled DNA in the final annealed product band divided by the total amount of DNA from all bands in the gel lane. All experiments were done in triplicate, and the error bars on each graph represent one S.D. in both directions. In most assays, 10-250 fmol of RPA was found to be sufficient for demonstrating strand annealing activities. At 50 fmol, RPA is approximately equimolar to the recombination intermediate complex because of all of the excess DNA needed to ensure that all (or almost all) of the labeled strand is in complex. Since each assay contained ∼50 fmol of single-stranded DNA, 100 fmol of RPA was used in all nontitration assays to maintain the 2:1 RPA/ssDNA ratio. We previously acquired evidence that RPA can accelerate the annealing of DNA strands (34Bartos J.D. Wang W. Pike J.E. Bambara R.A. J. Biol. Chem. 2006; 281: 32227-32239Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Our current work is an investigation of the conditions and substrate structures that support RPA-catalyzed strand annealing. Design of Oligonucleotide Substrates to Assess the Strand Annealing Properties of RPA—We first questioned whether the efficiency of RPA-catalyzed strand annealing correlates with the amount of structure in the single strands prior to annealing. We designed single-stranded oligomers with different folding structures that can anneal in various pairs (Table 1). The folding program mfold (version 3.2) was used to predict the free energy change (ΔG) for conversion of each strand from random coil to its lowest free energy folded structure. The hydrogen-bonding patterns of the lowest energy folding structures of those oligonucleotides are shown (Fig. 1). Oligomers with a positive ΔG are more likely to form a random coil than any consistent secondary structure and are thus designated as unstructured. Strands are designated as either templates (T) or complementary (C) strands, in which each C strand is complementary to an equal length or longer template. Two different templates were synthesized; T1 is a 57-nt oligomer that folds with a ΔG of -2.95 kcal/mol, and T2 is a 57-nt oligomer expected to remain a random coil based on a ΔG for folding of 4.2 kcal/mol. Two oligonucleotides that bind near the center of T1 were created. C5 is an 18-nt oligomer lacking secondary structure and is thus predicted to remain in the random coil configuration. Under the reaction conditions employed, it binds rapidly to T1 in the absence of RPA. Flap F1 contains the same 18 nt as C5 but has an additional 45 nt that are not complementary to T1. When F1 binds T1, the 45-nt region remains as a 3′ flap. However, when F1 is unannealed, folding is energetically favored with ΔG of -5.71 kcal/mol. RPA Can Promote Strand Annealing—The addition of RPA caused a progressive increase in the level of F1-T1 strand annealing as measured by the creation of the dsDNA complex (Fig. 2B). In the absence of RPA, F1 bound very slowly to T1 in our assay (Fig. 2A, lanes 1-5). Strand annealing in the absence of RPA was linear, with a rate of 0.026% of the radiolabeled template annealed/min over the entire 60 min. Linearity is anticipated, since the measurement was made under initial rate conditions in which the concentration of starting substrates does not change substantially during the course of the reaction. Annealing of C5 with T1, representing the same annealing sequence, but without structure, also occurred linearly, but at a rate of 0.934%/min, or 36 times faster than T1-F1 annealing (Fig. 2, C and D). These results demonstrate the inhibitory effect of secondary structure in the longer strand. They also show no evidence of spontaneous unannealing. The rate of F1-T1 annealing increased with the amount of RPA added, culminating in the highest rate of 0.452% of the radiolabeled template annealed/min over the first 15 min with 250 fmol of RPA, a greater than 17-fold increase (Fig. 2A, lanes 6-30). Evidence that RPA Strand Annealing Derives in Part from Its Strand Melting Properties—We compared the effect of RPA on the rate of annealing of template T1 to the structured flap F1 and to structureless complementary strand C5. A basal level of 31% of the labeled T1 DNA annealed to C5 during the 15-min period in the absence of RPA (Fig. 3, lane 1), whereas only 0.2% annealed to F1 (Fig. 3, lane 6). Titration of RPA lowered the annealed amount of the T1-C5 complex to 8.6% at 250 fmol, a decrease of 3.5-fold (Fig. 3, lanes 2-5). At the same time, titration of RPA raised the annealing level of the T1-F1 complex to 7.6% at 250 fmol, an increase of 3.5-fold (Fig. 3, lanes 7-10). This suggests that RPA strand annealing/melting reaches an equilibrium of ∼8% annealed for the 18-nt segment that is annealing. A reasonable interpretation of this result is that RPA is most effective at promoting the annealing of highly structured substrates, such as T1-F1, that would otherwise anneal at a very slow rate. However, RPA also has a strand melting activity that must be operating during the annealing reaction. T1 and C5 anneal rapidly in the absence of RPA, presumably because they have little interfering structure (see Fig. 1). However, 250 fmol of RPA is a very effective inhibitor of the rapid annealing reaction. It is notable that the level of annealing at 250 fmol of RPA becomes similar in both T1-F1 and T1-C5 annealing (Fig. 3B), and we found that RPA can unanneal both T1-F1 and T1-C5 preformed complexes (data not shown). The behavior of the system is consistent with the interpretation that RPA is displaying two activities, melting and annealing. When rapid spontaneous annealing is possible, the primary observable activity of RPA is melting. The amount of annealed strands at 250 fmol of RPA is reflective of the opposing reactions. RPA accelerated the poor annealing of the more structured strands (Fig. 2A, lanes 21-30). At high RPA, both strand pairs behaved similarly, consistent with the interpretation that RPA allows the structured strands to anneal as if they had little structure. This would occur if melting of the structure in the unannealed strands promoted the annealing reaction. Attempting to Simulate the Effects of RPA with Heat—We reasoned that if RPA promotes annealing through strand melting, increased temperature should have an equivalent effect on annealing; increased temperature should reduce the dependence of annealing on RPA, because heat can also destabilize structure that could interfere with annealing of single strands. The reaction temperature was increased for annealing of 57-nt T1 and 18-nt C5 in the absence of RPA (Fig. 4A). We found that RPA inhibited strand annealing of the 57-nt template and 18-nt complementary oligomer in the same manner regardless of increased temperature. Without RPA, the level of strand annealing increased with temperature, from 9.8% at 25 °C to 21.4% at 37 °C to 43.4% at 50 °C (Fig. 4A, lanes 1, 7, and 13). We interpreted the result to mean that the increased temperature destabilized intramolecular hydrogen bonds in T1, allowing it to bind C5. With this strand pair, the addition of RPA did not simulate the increase in temperature. The addition of RPA at 50 °C caused the level of strand annealing to decrease 13-fold, from 43.4% in the absence of RPA to 3.3% in the presence of 250 fmol of RPA. This suggests that RPA was either melting the dsDNA T1-C5 complex or preventing it from forming. RPA had a negative effect on annealing at both low and high concentration, showing that it is very effective at disrupting the T1-C5 complex (Fig. 4B). Clearly, with this strand pair, RPA and increased temperature had different effects, most likely because the highest temperature employed did not hinder annealing or melt the double-stranded product. Annealing of T1 and 57-nt C1 in the absence of RPA displayed a similar positive response to temperature. As with the T1-C5 annealing, the level of strand annealing rose more than 34-fold with the increase in temperature, from 1.9% at 25 °C to 65.6% at 50 °C (Fig. 4A, lanes 19 and 31). Unlike with the previous strand pair, the addition of RPA augmented the effect of the temperature increase, raising the amount of strand annealing to 9.5% at 25 °C and 81% at 50 °C. This suggests that RPA and increased temperature had additive effects on breaking the intramolecular hydrogen bonds that stabilize T1 and C1 secondary structure. However, unlike with the 18-nt C5, the addition of RPA increased strand annealing from 14.9% in the absence of RPA to 40% in the presence of 100 fmol of RPA at 37 °C. This implies that RPA and increased temperate collaborated in removing secondary structure from T1 and C1 to promote annealing. However, once T1 and C1 were annealed, RPA had difficulty melting the resulting 57 nt/57 nt dsDNA structure like it could the 18 nt/57 nt T1-C5 structure because of the additional 75-kcal/mol difference in free energy. The similar and additive effects of RPA and temperature with the longer strand are consistent with the interpretation that RPA promotes annealing by melting secondary structure in the reactant strands. We further compared effects of temperature and RPA by annealing flap F1 to T1. This strand pair utilized the same 18 nt that comprise C5 for annealing but with the addition of a 47-nt flap to give the C strand secondary structure (Fig. 4E). At 25 °C, the annealing pattern looked very similar to that which occurred using C1, suggesting that the secondary structure of the single strands initially inhibited annealing but the presence of RPA partially alleviated the interfering structure (Fig. 4F). Increasing the temperature from 25 to 37 °C raised annealing to a high of 10.5% at 100 fmol of RPA. A further increase in temperature shifted the annealing pattern higher, since in the absence of RPA the temperature is enough to relieve secondary structure. Interestingly, at 250 fmol of RPA, there was a reduction in annealing, reminiscent of the pattern seen using 18-nt C5; however, the resulting drop was much less than that seen with C5. Some nucleotides in the noncomplementary part of F1 can potentially form bonds with T1, contributing extra binding stability compared with the T1-C5 pair. We conclude that, as seen with C1, F1 secondary structure inhibited annealing until RPA or heat could reduce that structure. However, as seen with 18-nt C5 but not with 57-nt C1, the 18 nt did not anneal with enough stability to keep the resulting double-stranded product from being melted in the presence of high amounts of RPA. RPA Hinders the Annealing of Short Strands and Facilitates Annealing of Long Strands—For strand pairs that do not form flaps on annealing, the hypothesis that RPA displays both annealing and melting activities predicts that RPA would be most effective at catalyzing the annealing of a template strand to progressively longer complementary strands. This prediction is based on two expected properties of longer strands. First, the longer complementary (C) strand would anneal with greater stability to the template strand, resisting the melting activity of RPA (as suggested by data in Fig. 4, C and D). Moreover, the longer C strand would be more likely to have a greater negative free energy of folding, allowing it to respond more to the annealing function of the RPA. To test this idea, we measured the influence of RPA on the annealing rates of T1 to progressively longer co" @default.
• W1982181829 created "2016-06-24" @default.
• W1982181829 creator A5005315226 @default.
• W1982181829 creator A5008545738 @default.
• W1982181829 creator A5062158050 @default.
• W1982181829 creator A5069307927 @default.
• W1982181829 creator A5087322488 @default.
• W1982181829 date "2008-08-01" @default.
• W1982181829 modified "2023-10-18" @default.
• W1982181829 title "Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties" @default.
• W1982181829 cites W1485786260 @default.
• W1982181829 cites W1495312602 @default.
• W1982181829 cites W1550656265 @default.
• W1982181829 cites W1604305208 @default.
• W1982181829 cites W1605838085 @default.
• W1982181829 cites W1641947900 @default.
• W1982181829 cites W1818349386 @default.
• W1982181829 cites W1901653080 @default.
• W1982181829 cites W1965573008 @default.
• W1982181829 cites W1966219094 @default.
• W1982181829 cites W1972746487 @default.
• W1982181829 cites W1973544851 @default.
• W1982181829 cites W1974481952 @default.
• W1982181829 cites W1974823582 @default.
• W1982181829 cites W1989117798 @default.
• W1982181829 cites W1989821243 @default.
• W1982181829 cites W1991617089 @default.
• W1982181829 cites W1997002747 @default.
• W1982181829 cites W1997649177 @default.
• W1982181829 cites W1998607580 @default.
• W1982181829 cites W1999557018 @default.
• W1982181829 cites W1999660230 @default.
• W1982181829 cites W2010302368 @default.
• W1982181829 cites W2010544809 @default.
• W1982181829 cites W2017894593 @default.
• W1982181829 cites W2020143772 @default.
• W1982181829 cites W2030639168 @default.
• W1982181829 cites W2033891762 @default.
• W1982181829 cites W2034535098 @default.
• W1982181829 cites W2035746057 @default.
• W1982181829 cites W2041516645 @default.
• W1982181829 cites W2058654089 @default.
• W1982181829 cites W2059501883 @default.
• W1982181829 cites W2062043277 @default.
• W1982181829 cites W2064264662 @default.
• W1982181829 cites W2071098782 @default.
• W1982181829 cites W2076154132 @default.
• W1982181829 cites W2082129123 @default.
• W1982181829 cites W2082497259 @default.
• W1982181829 cites W2084164874 @default.
• W1982181829 cites W2090573596 @default.
• W1982181829 cites W2095206600 @default.
• W1982181829 cites W2107856790 @default.
• W1982181829 cites W2110161513 @default.
• W1982181829 cites W2110830902 @default.
• W1982181829 cites W2116538548 @default.
• W1982181829 cites W2123557271 @default.
• W1982181829 cites W2133573755 @default.
• W1982181829 cites W2135453449 @default.
• W1982181829 cites W2135995702 @default.
• W1982181829 cites W2139768549 @default.
• W1982181829 cites W2140528341 @default.
• W1982181829 cites W2140732239 @default.
• W1982181829 cites W2141157874 @default.
• W1982181829 cites W2144835937 @default.
• W1982181829 cites W2146851796 @default.
• W1982181829 cites W2154087197 @default.
• W1982181829 cites W2157811245 @default.
• W1982181829 cites W2161248815 @default.
• W1982181829 cites W2171775098 @default.
• W1982181829 doi "https://doi.org/10.1074/jbc.m800856200" @default.
• W1982181829 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2490778" @default.
• W1982181829 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18522944" @default.
• W1982181829 hasPublicationYear "2008" @default.
• W1982181829 type Work @default.
• W1982181829 sameAs 1982181829 @default.
• W1982181829 citedByCount "11" @default.
• W1982181829 countsByYear W19821818292013 @default.
• W1982181829 countsByYear W19821818292014 @default.
• W1982181829 countsByYear W19821818292016 @default.
• W1982181829 countsByYear W19821818292018 @default.
• W1982181829 countsByYear W19821818292020 @default.
• W1982181829 countsByYear W19821818292022 @default.
• W1982181829 crossrefType "journal-article" @default.
• W1982181829 hasAuthorship W1982181829A5005315226 @default.
• W1982181829 hasAuthorship W1982181829A5008545738 @default.
• W1982181829 hasAuthorship W1982181829A5062158050 @default.
• W1982181829 hasAuthorship W1982181829A5069307927 @default.
• W1982181829 hasAuthorship W1982181829A5087322488 @default.
• W1982181829 hasBestOaLocation W19821818291 @default.
• W1982181829 hasConcept C12554922 @default.
• W1982181829 hasConcept C12590798 @default.
• W1982181829 hasConcept C159047783 @default.
• W1982181829 hasConcept C185592680 @default.
• W1982181829 hasConcept C191897082 @default.
• W1982181829 hasConcept C192562407 @default.
• W1982181829 hasConcept C2777855556 @default.
• W1982181829 hasConcept C8010536 @default.

• next