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Yeast Pif1 accelerates annealing of complementary DNA strands.

Ramanagoudr-Bhojappa R, Byrd AK, Dahl C, Raney KD - Biochemistry (2014)

Bottom Line: We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex.Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+).Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205, United States.

ABSTRACT
Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+). Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3'-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

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Effect of varying ssDNA overhangs andPif1 concentrations on annealing.(a) DNA substrates that can anneal to form a 30bp duplex are shownwith the varying 5′- and 3′-overhangs indicated. Thesubstrates are named according to the length and type (5′ or3′) of ssDNA overhang and the length of duplex. They are shownin order from the most efficiently to least efficiently annealed.(b) Plot of the fraction of ssDNA annealed for the 30bp blunt endduplex (●), 20T 5′-overhang (■), 20T/20T fork(◆), 20T 3′-overhang (▲), 70T 5′-overhang(×), and 20T/20T dual 5′-overhangs (▼). Error barsrepresent the standard deviation of three independent experiments.Data were fit to Scheme 1, and the constantsfrom the fits are listed in Table 2. (c) Annealingof the 70T 5′-overhang product measured at 200, 100, 10, 2,0.4, and 0 nM Pif1, keeping the concentrations of the substrate strandsconstant at 2 nM radiolabeled 30nt CS and 2.6 nM 70T-30nt. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.9 × 107 M–1 s–1 and 0.022 s–1 for a 100:1 Pif1:DNA ratio, 1.9 × 107 M–1 s–1 and 0.037 s–1 for a 50:1 Pif1:DNA ratio, and 4.4 × 106 M–1 s–1 and 0.030 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed. (d) Annealing of the 30bp blunt endduplex under the same conditions described for panel c. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.5 × 107 M–1 s–1 and 0.14 s–1 for a 100:1 Pif1:DNA ratio, 1.5 × 107 M–1 s–1 and 0.14 s–1 for a 50:1 Pif1:DNA ratio, and 1.1 × 107 M–1 s–1 and 0.078 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed.
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fig4: Effect of varying ssDNA overhangs andPif1 concentrations on annealing.(a) DNA substrates that can anneal to form a 30bp duplex are shownwith the varying 5′- and 3′-overhangs indicated. Thesubstrates are named according to the length and type (5′ or3′) of ssDNA overhang and the length of duplex. They are shownin order from the most efficiently to least efficiently annealed.(b) Plot of the fraction of ssDNA annealed for the 30bp blunt endduplex (●), 20T 5′-overhang (■), 20T/20T fork(◆), 20T 3′-overhang (▲), 70T 5′-overhang(×), and 20T/20T dual 5′-overhangs (▼). Error barsrepresent the standard deviation of three independent experiments.Data were fit to Scheme 1, and the constantsfrom the fits are listed in Table 2. (c) Annealingof the 70T 5′-overhang product measured at 200, 100, 10, 2,0.4, and 0 nM Pif1, keeping the concentrations of the substrate strandsconstant at 2 nM radiolabeled 30nt CS and 2.6 nM 70T-30nt. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.9 × 107 M–1 s–1 and 0.022 s–1 for a 100:1 Pif1:DNA ratio, 1.9 × 107 M–1 s–1 and 0.037 s–1 for a 50:1 Pif1:DNA ratio, and 4.4 × 106 M–1 s–1 and 0.030 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed. (d) Annealing of the 30bp blunt endduplex under the same conditions described for panel c. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.5 × 107 M–1 s–1 and 0.14 s–1 for a 100:1 Pif1:DNA ratio, 1.5 × 107 M–1 s–1 and 0.14 s–1 for a 50:1 Pif1:DNA ratio, and 1.1 × 107 M–1 s–1 and 0.078 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed.

Mentions: The effectof changes in the length and type of ssDNA overhangs, such as a shortoverhang versus a long overhang, a 3′-overhang versus a 5′-overhang,and one versus two overhangs, on Pif1-promoted strand annealing wasinvestigated. Several substrates (Table 1)that would generate a 30bp duplex product (Figure 4a) with varying length overhangs were tested in the absenceof ATP and Mg2+ to eliminate complicating effects of unwinding(Figure 4b). Data were fit to the annealingmechanism in Scheme 1. The constants obtainedfrom the fit are listed in Table 2. The second-orderrate constant for annealing is similar for each of the substrate pairs.What varies between the substrates is the fraction of substrate thatis available for annealing, indicated by the rate constant for conversionof S1 to S, where S is substrate that is in a conformation that isnot amenable to annealing. (A similar process could occur with S2.However, we included this only for one of the substrate strands tolimit the number of variables in the fit and use the simplest mechanismpossible to fit the data.) S could be some conformation in which thebases are not fully exposed because of short regions of base pairingwithin the substrate or the potential sequestration of bases by anexcess of Pif1 bound to the substrate. A modest difference in thefraction of ssDNA annealed is observed for the products containinga 5′-overhang or 3′-overhang, with the 5′-overhangbeing the preferred product. In general, longer substrates are preferred(Figure 4b), but the length is not the onlyfactor that influences annealing. Two different products, each containingtwo 20nt ssDNA overhangs, were compared. The forked duplex (cyan diamonds)has a ssDNA overhang on the 5′-end of one strand and the 3′-endof the other. The dual 5′-overhang product has 20nt ssDNA overhangson each 5′-end (black triangles). The dual 5′-overhangproduct is formed preferentially over the forked product even thoughthe total length of the substrates and overhangs is equivalent, againindicating that 5′-overhangs are preferred. The dual-5′-overhangproduct (two 20T overhangs) is annealed similarly to a product witha single 70T 5′-overhang even though the total length is reduced,indicating that the location of overhangs is important, in additionto total length. Although the ratio of Pif1 to DNA strands is thesame for each of the substrate pairs utilized, the ratio of Pif1 tobinding sites varies significantly between the substrate pairs dueto the variation in substrate length. We note that the substrate preferencereported at saturating enzyme concentrations may not be applicablewhen the enzyme concentration is not saturating because of the differencein the number of binding sites.


Yeast Pif1 accelerates annealing of complementary DNA strands.

Ramanagoudr-Bhojappa R, Byrd AK, Dahl C, Raney KD - Biochemistry (2014)

Effect of varying ssDNA overhangs andPif1 concentrations on annealing.(a) DNA substrates that can anneal to form a 30bp duplex are shownwith the varying 5′- and 3′-overhangs indicated. Thesubstrates are named according to the length and type (5′ or3′) of ssDNA overhang and the length of duplex. They are shownin order from the most efficiently to least efficiently annealed.(b) Plot of the fraction of ssDNA annealed for the 30bp blunt endduplex (●), 20T 5′-overhang (■), 20T/20T fork(◆), 20T 3′-overhang (▲), 70T 5′-overhang(×), and 20T/20T dual 5′-overhangs (▼). Error barsrepresent the standard deviation of three independent experiments.Data were fit to Scheme 1, and the constantsfrom the fits are listed in Table 2. (c) Annealingof the 70T 5′-overhang product measured at 200, 100, 10, 2,0.4, and 0 nM Pif1, keeping the concentrations of the substrate strandsconstant at 2 nM radiolabeled 30nt CS and 2.6 nM 70T-30nt. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.9 × 107 M–1 s–1 and 0.022 s–1 for a 100:1 Pif1:DNA ratio, 1.9 × 107 M–1 s–1 and 0.037 s–1 for a 50:1 Pif1:DNA ratio, and 4.4 × 106 M–1 s–1 and 0.030 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed. (d) Annealing of the 30bp blunt endduplex under the same conditions described for panel c. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.5 × 107 M–1 s–1 and 0.14 s–1 for a 100:1 Pif1:DNA ratio, 1.5 × 107 M–1 s–1 and 0.14 s–1 for a 50:1 Pif1:DNA ratio, and 1.1 × 107 M–1 s–1 and 0.078 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed.
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fig4: Effect of varying ssDNA overhangs andPif1 concentrations on annealing.(a) DNA substrates that can anneal to form a 30bp duplex are shownwith the varying 5′- and 3′-overhangs indicated. Thesubstrates are named according to the length and type (5′ or3′) of ssDNA overhang and the length of duplex. They are shownin order from the most efficiently to least efficiently annealed.(b) Plot of the fraction of ssDNA annealed for the 30bp blunt endduplex (●), 20T 5′-overhang (■), 20T/20T fork(◆), 20T 3′-overhang (▲), 70T 5′-overhang(×), and 20T/20T dual 5′-overhangs (▼). Error barsrepresent the standard deviation of three independent experiments.Data were fit to Scheme 1, and the constantsfrom the fits are listed in Table 2. (c) Annealingof the 70T 5′-overhang product measured at 200, 100, 10, 2,0.4, and 0 nM Pif1, keeping the concentrations of the substrate strandsconstant at 2 nM radiolabeled 30nt CS and 2.6 nM 70T-30nt. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.9 × 107 M–1 s–1 and 0.022 s–1 for a 100:1 Pif1:DNA ratio, 1.9 × 107 M–1 s–1 and 0.037 s–1 for a 50:1 Pif1:DNA ratio, and 4.4 × 106 M–1 s–1 and 0.030 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed. (d) Annealing of the 30bp blunt endduplex under the same conditions described for panel c. Duplicateexperiments produced similar results. Data were fit to Scheme 1 to obtain the second-order rate constant for annealingand the rate constant for conversion of S1 to S (1.5 × 107 M–1 s–1 and 0.14 s–1 for a 100:1 Pif1:DNA ratio, 1.5 × 107 M–1 s–1 and 0.14 s–1 for a 50:1 Pif1:DNA ratio, and 1.1 × 107 M–1 s–1 and 0.078 s–1 for a 5:1Pif1:DNA ratio, respectively). Data obtained at 1:1 and 1:5 Pif1:DNAratios and in the absence of Pif1 were not fit because of the smallquantities of product formed.
Mentions: The effectof changes in the length and type of ssDNA overhangs, such as a shortoverhang versus a long overhang, a 3′-overhang versus a 5′-overhang,and one versus two overhangs, on Pif1-promoted strand annealing wasinvestigated. Several substrates (Table 1)that would generate a 30bp duplex product (Figure 4a) with varying length overhangs were tested in the absenceof ATP and Mg2+ to eliminate complicating effects of unwinding(Figure 4b). Data were fit to the annealingmechanism in Scheme 1. The constants obtainedfrom the fit are listed in Table 2. The second-orderrate constant for annealing is similar for each of the substrate pairs.What varies between the substrates is the fraction of substrate thatis available for annealing, indicated by the rate constant for conversionof S1 to S, where S is substrate that is in a conformation that isnot amenable to annealing. (A similar process could occur with S2.However, we included this only for one of the substrate strands tolimit the number of variables in the fit and use the simplest mechanismpossible to fit the data.) S could be some conformation in which thebases are not fully exposed because of short regions of base pairingwithin the substrate or the potential sequestration of bases by anexcess of Pif1 bound to the substrate. A modest difference in thefraction of ssDNA annealed is observed for the products containinga 5′-overhang or 3′-overhang, with the 5′-overhangbeing the preferred product. In general, longer substrates are preferred(Figure 4b), but the length is not the onlyfactor that influences annealing. Two different products, each containingtwo 20nt ssDNA overhangs, were compared. The forked duplex (cyan diamonds)has a ssDNA overhang on the 5′-end of one strand and the 3′-endof the other. The dual 5′-overhang product has 20nt ssDNA overhangson each 5′-end (black triangles). The dual 5′-overhangproduct is formed preferentially over the forked product even thoughthe total length of the substrates and overhangs is equivalent, againindicating that 5′-overhangs are preferred. The dual-5′-overhangproduct (two 20T overhangs) is annealed similarly to a product witha single 70T 5′-overhang even though the total length is reduced,indicating that the location of overhangs is important, in additionto total length. Although the ratio of Pif1 to DNA strands is thesame for each of the substrate pairs utilized, the ratio of Pif1 tobinding sites varies significantly between the substrate pairs dueto the variation in substrate length. We note that the substrate preferencereported at saturating enzyme concentrations may not be applicablewhen the enzyme concentration is not saturating because of the differencein the number of binding sites.

Bottom Line: We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex.Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+).Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205, United States.

ABSTRACT
Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+). Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3'-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

Show MeSH
Related in: MedlinePlus