Limits...
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.

Show MeSH

Related in: MedlinePlus

Pif1 exhibited robuststrand annealing activity in the absenceof ATP and MgCl2. (a) Schematic illustration for two partiallycomplementary ssDNAs (70T-30nt and 30nt CS) annealing to generatea partial duplex DNA, 70T-30bp. Pif1 (200 nM) was incubated with 70T-30nt(2.6 nM) and 30nt CS (2 nM) for increasing times, and the reactionswere stopped by mixing with a quench solution containing 200 mM EDTA,1% SDS, 100 nM DNA trap, and 5 mg/mL dextran sulfate. (b) Representativegel images of annealed products formed in the presence of Pif1 (top)and in the absence of Pif1 (bottom) as a function of time. (c) Fractionof ssDNA annealed in the presence of Pif1 (circles). Spontaneous annealingwas measured in the absence of Pif1 (diamonds). Error bars representthe standard deviation of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263423&req=5

fig2: Pif1 exhibited robuststrand annealing activity in the absenceof ATP and MgCl2. (a) Schematic illustration for two partiallycomplementary ssDNAs (70T-30nt and 30nt CS) annealing to generatea partial duplex DNA, 70T-30bp. Pif1 (200 nM) was incubated with 70T-30nt(2.6 nM) and 30nt CS (2 nM) for increasing times, and the reactionswere stopped by mixing with a quench solution containing 200 mM EDTA,1% SDS, 100 nM DNA trap, and 5 mg/mL dextran sulfate. (b) Representativegel images of annealed products formed in the presence of Pif1 (top)and in the absence of Pif1 (bottom) as a function of time. (c) Fractionof ssDNA annealed in the presence of Pif1 (circles). Spontaneous annealingwas measured in the absence of Pif1 (diamonds). Error bars representthe standard deviation of three independent experiments.

Mentions: Pif1’s abilityto anneal complementary strands was initially investigated in theabsence of ATP and Mg2+ to eliminate the effects of unwinding.A pair of complementary ssDNA substrates that would generate a partialduplex product were used to test Pif1’s ability to anneal (Figure 2a). The annealed products were separated from ssDNAon a native polyacrylamide gel (Figure 2b).Annealing of 70T-30nt with a 30nt complementary strand generates a70T-30bp product (Table 1). Approximately 80%of the 70T-30bp product was formed in <2 min, whereas no productformation was observed in the absence of the enzyme during the timeperiod of the reaction (Figure 2c), indicatingPif1 can promote annealing of complementary strands.


Yeast Pif1 accelerates annealing of complementary DNA strands.

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

Pif1 exhibited robuststrand annealing activity in the absenceof ATP and MgCl2. (a) Schematic illustration for two partiallycomplementary ssDNAs (70T-30nt and 30nt CS) annealing to generatea partial duplex DNA, 70T-30bp. Pif1 (200 nM) was incubated with 70T-30nt(2.6 nM) and 30nt CS (2 nM) for increasing times, and the reactionswere stopped by mixing with a quench solution containing 200 mM EDTA,1% SDS, 100 nM DNA trap, and 5 mg/mL dextran sulfate. (b) Representativegel images of annealed products formed in the presence of Pif1 (top)and in the absence of Pif1 (bottom) as a function of time. (c) Fractionof ssDNA annealed in the presence of Pif1 (circles). Spontaneous annealingwas measured in the absence of Pif1 (diamonds). Error bars representthe standard deviation of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263423&req=5

fig2: Pif1 exhibited robuststrand annealing activity in the absenceof ATP and MgCl2. (a) Schematic illustration for two partiallycomplementary ssDNAs (70T-30nt and 30nt CS) annealing to generatea partial duplex DNA, 70T-30bp. Pif1 (200 nM) was incubated with 70T-30nt(2.6 nM) and 30nt CS (2 nM) for increasing times, and the reactionswere stopped by mixing with a quench solution containing 200 mM EDTA,1% SDS, 100 nM DNA trap, and 5 mg/mL dextran sulfate. (b) Representativegel images of annealed products formed in the presence of Pif1 (top)and in the absence of Pif1 (bottom) as a function of time. (c) Fractionof ssDNA annealed in the presence of Pif1 (circles). Spontaneous annealingwas measured in the absence of Pif1 (diamonds). Error bars representthe standard deviation of three independent experiments.
Mentions: Pif1’s abilityto anneal complementary strands was initially investigated in theabsence of ATP and Mg2+ to eliminate the effects of unwinding.A pair of complementary ssDNA substrates that would generate a partialduplex product were used to test Pif1’s ability to anneal (Figure 2a). The annealed products were separated from ssDNAon a native polyacrylamide gel (Figure 2b).Annealing of 70T-30nt with a 30nt complementary strand generates a70T-30bp product (Table 1). Approximately 80%of the 70T-30bp product was formed in <2 min, whereas no productformation was observed in the absence of the enzyme during the timeperiod of the reaction (Figure 2c), indicatingPif1 can promote annealing of complementary strands.

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