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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-catalyzed unwinding yielded differential productappearancein the presence or absence of DNA trap. (a) Duplex DNA substrate wasmixed with Pif1 in the presence of ATP and MgCl2 to formssDNA product. A DNA trap complementary to the unlabeled strand wasincluded in some reactions. (b) Unwinding experiments were initiatedby mixing 2 nM 70T-30bp substrate, ATP, and MgCl2 with200 nM Pif1. Reactions were performed either in the absence (triangles)or in the presence (squares) of unlabeled 60 nM DNA trap. Fittingthe data to the equation for a single exponential resulted in unwindingrate constants of 0.80 ± 0.02 and 1.3 ± 0.04 min–1 for unwinding in the presence and absence of DNA trap, respectively.The amplitudes of the product formation curves are 0.87 ± 0.01and 0.56 ± 0.01 for unwinding in the presence and absence ofa DNA trap, respectively.
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fig1: Pif1-catalyzed unwinding yielded differential productappearancein the presence or absence of DNA trap. (a) Duplex DNA substrate wasmixed with Pif1 in the presence of ATP and MgCl2 to formssDNA product. A DNA trap complementary to the unlabeled strand wasincluded in some reactions. (b) Unwinding experiments were initiatedby mixing 2 nM 70T-30bp substrate, ATP, and MgCl2 with200 nM Pif1. Reactions were performed either in the absence (triangles)or in the presence (squares) of unlabeled 60 nM DNA trap. Fittingthe data to the equation for a single exponential resulted in unwindingrate constants of 0.80 ± 0.02 and 1.3 ± 0.04 min–1 for unwinding in the presence and absence of DNA trap, respectively.The amplitudes of the product formation curves are 0.87 ± 0.01and 0.56 ± 0.01 for unwinding in the presence and absence ofa DNA trap, respectively.

Mentions: A likely role forPif1 in promoting strand annealing activity was identified when anunwinding experiment showed a disparity in product formation withor without a DNA trap (Figure 1). Pif1-catalyzedunwinding experiments were performed in the presence or absence ofa DNA trap that captures the unlabeled displaced strand, leaving theunwound radiolabeled strand as the ssDNA product. Less ssDNA was observedfrom Pif1-catalyzed unwinding of the 70T-30bp substrate when the DNAtrap was not included (Figure 1b). In the absenceof a DNA trap, the amount of ssDNA generated reached a plateau at∼50%, suggesting that the unwound products were reannealedand an equilibrium had been reached between unwinding and reannealingactivities, as has been shown previously for WRN and BLM helicases.48 These results suggest that Pif1 might promotestrand annealing.


Yeast Pif1 accelerates annealing of complementary DNA strands.

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

Pif1-catalyzed unwinding yielded differential productappearancein the presence or absence of DNA trap. (a) Duplex DNA substrate wasmixed with Pif1 in the presence of ATP and MgCl2 to formssDNA product. A DNA trap complementary to the unlabeled strand wasincluded in some reactions. (b) Unwinding experiments were initiatedby mixing 2 nM 70T-30bp substrate, ATP, and MgCl2 with200 nM Pif1. Reactions were performed either in the absence (triangles)or in the presence (squares) of unlabeled 60 nM DNA trap. Fittingthe data to the equation for a single exponential resulted in unwindingrate constants of 0.80 ± 0.02 and 1.3 ± 0.04 min–1 for unwinding in the presence and absence of DNA trap, respectively.The amplitudes of the product formation curves are 0.87 ± 0.01and 0.56 ± 0.01 for unwinding in the presence and absence ofa DNA trap, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Pif1-catalyzed unwinding yielded differential productappearancein the presence or absence of DNA trap. (a) Duplex DNA substrate wasmixed with Pif1 in the presence of ATP and MgCl2 to formssDNA product. A DNA trap complementary to the unlabeled strand wasincluded in some reactions. (b) Unwinding experiments were initiatedby mixing 2 nM 70T-30bp substrate, ATP, and MgCl2 with200 nM Pif1. Reactions were performed either in the absence (triangles)or in the presence (squares) of unlabeled 60 nM DNA trap. Fittingthe data to the equation for a single exponential resulted in unwindingrate constants of 0.80 ± 0.02 and 1.3 ± 0.04 min–1 for unwinding in the presence and absence of DNA trap, respectively.The amplitudes of the product formation curves are 0.87 ± 0.01and 0.56 ± 0.01 for unwinding in the presence and absence ofa DNA trap, respectively.
Mentions: A likely role forPif1 in promoting strand annealing activity was identified when anunwinding experiment showed a disparity in product formation withor without a DNA trap (Figure 1). Pif1-catalyzedunwinding experiments were performed in the presence or absence ofa DNA trap that captures the unlabeled displaced strand, leaving theunwound radiolabeled strand as the ssDNA product. Less ssDNA was observedfrom Pif1-catalyzed unwinding of the 70T-30bp substrate when the DNAtrap was not included (Figure 1b). In the absenceof a DNA trap, the amount of ssDNA generated reached a plateau at∼50%, suggesting that the unwound products were reannealedand an equilibrium had been reached between unwinding and reannealingactivities, as has been shown previously for WRN and BLM helicases.48 These results suggest that Pif1 might promotestrand 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