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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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Pif1-catalyzedstrand annealing using longer DNA substrates. (a)Schematic illustration for strand annealing experiments using 80ntstrand and 80nt CS to generate an 80bp blunt end dsDNA product. (b)Results for Pif1 (200 nM) annealing of the 80nt strand (2.6 nM) withradiolabeled 80nt CS (2 nM) to generate an 80bp blunt DNA productin the presence of ATP and MgCl2 (red circles). Annealingby Pif1 in the absence of ATP but in the presence of MgCl2 is shown as green squares. Spontaneous annealing in the absenceof enzyme in the presence of ATP and MgCl2 was also measured(blue diamonds). Data were fit to a simple annealing mechanism (Scheme 2) to obtain second-order rate constants for annealingof 3.0 × 106 M–1 s–1 in the presence of Pif1 and ATP, 2.9 × 107 M–1 s–1 in the presence of Pif1 butin the absence of ATP, and 3.0 × 105 M–1 s–1 in the absence of Pif1.
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fig6: Pif1-catalyzedstrand annealing using longer DNA substrates. (a)Schematic illustration for strand annealing experiments using 80ntstrand and 80nt CS to generate an 80bp blunt end dsDNA product. (b)Results for Pif1 (200 nM) annealing of the 80nt strand (2.6 nM) withradiolabeled 80nt CS (2 nM) to generate an 80bp blunt DNA productin the presence of ATP and MgCl2 (red circles). Annealingby Pif1 in the absence of ATP but in the presence of MgCl2 is shown as green squares. Spontaneous annealing in the absenceof enzyme in the presence of ATP and MgCl2 was also measured(blue diamonds). Data were fit to a simple annealing mechanism (Scheme 2) to obtain second-order rate constants for annealingof 3.0 × 106 M–1 s–1 in the presence of Pif1 and ATP, 2.9 × 107 M–1 s–1 in the presence of Pif1 butin the absence of ATP, and 3.0 × 105 M–1 s–1 in the absence of Pif1.

Mentions: Pif1-promotedstrand annealing experiments in the presence of ATP and MgCl2 using 70T-30bp (Figure 5) product showedthat the unwinding and annealing activities of Pif1 reached equilibrium.It is well-established that Pif1 has relatively low processivity forunwinding,17,19 suggesting that an increase induplex length should shift the equilibrium toward annealing. In addition,substrates that would generate an annealed product that is a bluntend duplex or has a 3′-ssDNA overhang should produce more productin an annealing reaction because the product should not be unwoundby the 5′-to-3′ helicase activity of Pif1. To test this,two complementary ssDNAs that can generate an 80bp blunt DNA productwere used (Figure 6a). Pif1 strand annealingactivity in the presence of ATP and MgCl2 led to nearlycomplete annealing of the substrates (Figure 6b) relative to annealing of substrates that produce a product thatcould be readily unwound (Figure 5). The rateconstant for annealing based on a fit of the data to a simple annealingmechanism (Scheme 2) is 3.0 × 106 M–1 s–1. When ATP was absent,the second-order rate constant for annealing was 2.9 × 107 M–1 s–1. Faster annealingin the absence of ATP suggests that even though the final productof the annealing reaction is not a substrate for Pif1 unwinding, apartially annealed intermediate could be a substrate for unwindingby Pif1. Spontaneous strand annealing was observed for formation ofthe 80bp blunt DNA product, with a rate constant of 3.0 × 105 M–1 s–1. This is similarto previous DNA hybridization rate measurements of 2.0 × 105 to 1.2 × 106 M–1 s–1, depending on the sequence.51 However, annealing by Pif1 was 10-fold faster than spontaneous annealing.


Yeast Pif1 accelerates annealing of complementary DNA strands.

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

Pif1-catalyzedstrand annealing using longer DNA substrates. (a)Schematic illustration for strand annealing experiments using 80ntstrand and 80nt CS to generate an 80bp blunt end dsDNA product. (b)Results for Pif1 (200 nM) annealing of the 80nt strand (2.6 nM) withradiolabeled 80nt CS (2 nM) to generate an 80bp blunt DNA productin the presence of ATP and MgCl2 (red circles). Annealingby Pif1 in the absence of ATP but in the presence of MgCl2 is shown as green squares. Spontaneous annealing in the absenceof enzyme in the presence of ATP and MgCl2 was also measured(blue diamonds). Data were fit to a simple annealing mechanism (Scheme 2) to obtain second-order rate constants for annealingof 3.0 × 106 M–1 s–1 in the presence of Pif1 and ATP, 2.9 × 107 M–1 s–1 in the presence of Pif1 butin the absence of ATP, and 3.0 × 105 M–1 s–1 in the absence of Pif1.
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Related In: Results  -  Collection

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fig6: Pif1-catalyzedstrand annealing using longer DNA substrates. (a)Schematic illustration for strand annealing experiments using 80ntstrand and 80nt CS to generate an 80bp blunt end dsDNA product. (b)Results for Pif1 (200 nM) annealing of the 80nt strand (2.6 nM) withradiolabeled 80nt CS (2 nM) to generate an 80bp blunt DNA productin the presence of ATP and MgCl2 (red circles). Annealingby Pif1 in the absence of ATP but in the presence of MgCl2 is shown as green squares. Spontaneous annealing in the absenceof enzyme in the presence of ATP and MgCl2 was also measured(blue diamonds). Data were fit to a simple annealing mechanism (Scheme 2) to obtain second-order rate constants for annealingof 3.0 × 106 M–1 s–1 in the presence of Pif1 and ATP, 2.9 × 107 M–1 s–1 in the presence of Pif1 butin the absence of ATP, and 3.0 × 105 M–1 s–1 in the absence of Pif1.
Mentions: Pif1-promotedstrand annealing experiments in the presence of ATP and MgCl2 using 70T-30bp (Figure 5) product showedthat the unwinding and annealing activities of Pif1 reached equilibrium.It is well-established that Pif1 has relatively low processivity forunwinding,17,19 suggesting that an increase induplex length should shift the equilibrium toward annealing. In addition,substrates that would generate an annealed product that is a bluntend duplex or has a 3′-ssDNA overhang should produce more productin an annealing reaction because the product should not be unwoundby the 5′-to-3′ helicase activity of Pif1. To test this,two complementary ssDNAs that can generate an 80bp blunt DNA productwere used (Figure 6a). Pif1 strand annealingactivity in the presence of ATP and MgCl2 led to nearlycomplete annealing of the substrates (Figure 6b) relative to annealing of substrates that produce a product thatcould be readily unwound (Figure 5). The rateconstant for annealing based on a fit of the data to a simple annealingmechanism (Scheme 2) is 3.0 × 106 M–1 s–1. When ATP was absent,the second-order rate constant for annealing was 2.9 × 107 M–1 s–1. Faster annealingin the absence of ATP suggests that even though the final productof the annealing reaction is not a substrate for Pif1 unwinding, apartially annealed intermediate could be a substrate for unwindingby Pif1. Spontaneous strand annealing was observed for formation ofthe 80bp blunt DNA product, with a rate constant of 3.0 × 105 M–1 s–1. This is similarto previous DNA hybridization rate measurements of 2.0 × 105 to 1.2 × 106 M–1 s–1, depending on the sequence.51 However, annealing by Pif1 was 10-fold faster than spontaneous annealing.

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