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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 ATP and Mg2+ on Pif1 strand annealing activity.Shown are the kinetic plots observed for Pif1 (200 nM) catalyzed annealingof 70T-30nt (2 nM) and 30nt CS (2.6 nM) to generate 70T-30bp in thepresence (blue) or absence (red) of 5 mM ATP. Control reaction mixturesin the presence (green) or absence (black) of ATP lacked Pif1. Allreaction mixtures contained 10 mM MgCl2. Data collectedin the presence of ATP and Pif1 were fit to Scheme 3 to obtain a second-order rate constant of (5.1 ± 0.3)× 106 M–1 s–1 forannealing and a rate constant of 0.021 ± 0.004 s–1 for unwinding. Data collected in the absence of ATP but in the presenceof Pif1 were fit to Scheme 1, and a second-orderrate constant of (1.3 ± 0.2) × 107 M–1 s–1 for annealing and a rate constant of 0.094± 0.004 s–1 for conversion between S1 and Swere obtained. In the absence of Pif1, data were fit to Scheme 2, and the second-order rate constants for annealingwere (1.7 ± 0.9) × 105 and (5.8 ± 0.3) ×105 M–1 s–1 in thepresence and absence of ATP, respectively.
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fig5: Effect of ATP and Mg2+ on Pif1 strand annealing activity.Shown are the kinetic plots observed for Pif1 (200 nM) catalyzed annealingof 70T-30nt (2 nM) and 30nt CS (2.6 nM) to generate 70T-30bp in thepresence (blue) or absence (red) of 5 mM ATP. Control reaction mixturesin the presence (green) or absence (black) of ATP lacked Pif1. Allreaction mixtures contained 10 mM MgCl2. Data collectedin the presence of ATP and Pif1 were fit to Scheme 3 to obtain a second-order rate constant of (5.1 ± 0.3)× 106 M–1 s–1 forannealing and a rate constant of 0.021 ± 0.004 s–1 for unwinding. Data collected in the absence of ATP but in the presenceof Pif1 were fit to Scheme 1, and a second-orderrate constant of (1.3 ± 0.2) × 107 M–1 s–1 for annealing and a rate constant of 0.094± 0.004 s–1 for conversion between S1 and Swere obtained. In the absence of Pif1, data were fit to Scheme 2, and the second-order rate constants for annealingwere (1.7 ± 0.9) × 105 and (5.8 ± 0.3) ×105 M–1 s–1 in thepresence and absence of ATP, respectively.

Mentions: Helicases hydrolyze ATP, so the strand annealingactivity was examinedunder conditions in which strand separation can also occur. Annealingby Pif1 was tested in the presence or absence of ATP in the presenceof MgCl2 using the 70T-30bp product (Figure 5). After the initial rapid phase, the level of product formationreached a plateau, indicating that unwinding and annealing were atequilibrium. This is similar to previous observations with Dda helicasein which the annealing product serves as a substrate for strand separation.43 Data collected in the presence of ATP and Pif1were fit to Scheme 3 to obtain a second-orderrate constant of (5.1 ± 0.3) × 106 M–1 s–1 for annealing and a rate constant of 0.021± 0.004 s–1 for unwinding. Data collected inthe absence of ATP but in the presence of Pif1 were fit to Scheme 1, and a second-order rate constant of (1.3 ±0.2) × 107 M–1 s–1 for annealing was obtained. In the absence of Pif1, data were fitto Scheme 2, and the second-order rate constantsfor annealing were (1.7 ± 0.9) × 105 and (5.8± 0.3) × 105 M–1 s–1 in the presence and absence of ATP, respectively. The reduced spontaneousannealing rate in the presence of ATP is likely due to sequestrationof Mg2+ by ATP, thereby weakening the ability of Mg2+ to stabilize the duplex. Notably, Pif1 enhances the spontaneousannealing rate 20-fold in the absence of ATP and 30-fold in the presenceof ATP.


Yeast Pif1 accelerates annealing of complementary DNA strands.

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

Effect of ATP and Mg2+ on Pif1 strand annealing activity.Shown are the kinetic plots observed for Pif1 (200 nM) catalyzed annealingof 70T-30nt (2 nM) and 30nt CS (2.6 nM) to generate 70T-30bp in thepresence (blue) or absence (red) of 5 mM ATP. Control reaction mixturesin the presence (green) or absence (black) of ATP lacked Pif1. Allreaction mixtures contained 10 mM MgCl2. Data collectedin the presence of ATP and Pif1 were fit to Scheme 3 to obtain a second-order rate constant of (5.1 ± 0.3)× 106 M–1 s–1 forannealing and a rate constant of 0.021 ± 0.004 s–1 for unwinding. Data collected in the absence of ATP but in the presenceof Pif1 were fit to Scheme 1, and a second-orderrate constant of (1.3 ± 0.2) × 107 M–1 s–1 for annealing and a rate constant of 0.094± 0.004 s–1 for conversion between S1 and Swere obtained. In the absence of Pif1, data were fit to Scheme 2, and the second-order rate constants for annealingwere (1.7 ± 0.9) × 105 and (5.8 ± 0.3) ×105 M–1 s–1 in thepresence and absence of ATP, respectively.
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Related In: Results  -  Collection

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fig5: Effect of ATP and Mg2+ on Pif1 strand annealing activity.Shown are the kinetic plots observed for Pif1 (200 nM) catalyzed annealingof 70T-30nt (2 nM) and 30nt CS (2.6 nM) to generate 70T-30bp in thepresence (blue) or absence (red) of 5 mM ATP. Control reaction mixturesin the presence (green) or absence (black) of ATP lacked Pif1. Allreaction mixtures contained 10 mM MgCl2. Data collectedin the presence of ATP and Pif1 were fit to Scheme 3 to obtain a second-order rate constant of (5.1 ± 0.3)× 106 M–1 s–1 forannealing and a rate constant of 0.021 ± 0.004 s–1 for unwinding. Data collected in the absence of ATP but in the presenceof Pif1 were fit to Scheme 1, and a second-orderrate constant of (1.3 ± 0.2) × 107 M–1 s–1 for annealing and a rate constant of 0.094± 0.004 s–1 for conversion between S1 and Swere obtained. In the absence of Pif1, data were fit to Scheme 2, and the second-order rate constants for annealingwere (1.7 ± 0.9) × 105 and (5.8 ± 0.3) ×105 M–1 s–1 in thepresence and absence of ATP, respectively.
Mentions: Helicases hydrolyze ATP, so the strand annealingactivity was examinedunder conditions in which strand separation can also occur. Annealingby Pif1 was tested in the presence or absence of ATP in the presenceof MgCl2 using the 70T-30bp product (Figure 5). After the initial rapid phase, the level of product formationreached a plateau, indicating that unwinding and annealing were atequilibrium. This is similar to previous observations with Dda helicasein which the annealing product serves as a substrate for strand separation.43 Data collected in the presence of ATP and Pif1were fit to Scheme 3 to obtain a second-orderrate constant of (5.1 ± 0.3) × 106 M–1 s–1 for annealing and a rate constant of 0.021± 0.004 s–1 for unwinding. Data collected inthe absence of ATP but in the presence of Pif1 were fit to Scheme 1, and a second-order rate constant of (1.3 ±0.2) × 107 M–1 s–1 for annealing was obtained. In the absence of Pif1, data were fitto Scheme 2, and the second-order rate constantsfor annealing were (1.7 ± 0.9) × 105 and (5.8± 0.3) × 105 M–1 s–1 in the presence and absence of ATP, respectively. The reduced spontaneousannealing rate in the presence of ATP is likely due to sequestrationof Mg2+ by ATP, thereby weakening the ability of Mg2+ to stabilize the duplex. Notably, Pif1 enhances the spontaneousannealing rate 20-fold in the absence of ATP and 30-fold in the presenceof ATP.

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