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
Pif1 can bind multiple strands of DNA. (a) Schematic illustrationof fluorescence titration. Two noncomplementary 8-mer oligonucleotides(100 nM) were mixed and then titrated with Pif1. Fluorescence emissionwas measured at 668 nm with excitation at 550 nm. (b) Increase inCy5 fluorescence emission as a function of Pif1 concentration (blue).Data were fit to the Hill equation to obtain the concentration ofprotein at which half of the maximal FRET change is observed, 200nM Pif1, and the Hill coefficient of 2.1. Duplicate experiments producedsimilar results. No increase in fluorescence was observed in controlreactions in which Pif1 was added to only the Cy3-labeled oligonucleotide(red) or only the Cy5-labeled oligonucleotide (black).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Pif1 can bind multiple strands of DNA. (a) Schematic illustrationof fluorescence titration. Two noncomplementary 8-mer oligonucleotides(100 nM) were mixed and then titrated with Pif1. Fluorescence emissionwas measured at 668 nm with excitation at 550 nm. (b) Increase inCy5 fluorescence emission as a function of Pif1 concentration (blue).Data were fit to the Hill equation to obtain the concentration ofprotein at which half of the maximal FRET change is observed, 200nM Pif1, and the Hill coefficient of 2.1. Duplicate experiments producedsimilar results. No increase in fluorescence was observed in controlreactions in which Pif1 was added to only the Cy3-labeled oligonucleotide(red) or only the Cy5-labeled oligonucleotide (black).

Mentions: A FRET binding experiment was designed to investigate the abilityof Pif1 to simultaneously bind multiple DNA strands (Figure 3a). Like other superfamily 1 helicases, Pif1 sequestersapproximately eight nucleotides on the basis of a repeating patternof nucleotide protection of approximately eight nucleotides in thepresence of Pif1 in KMnO4 footprinting experiments (FigureS1 of the Supporting Information), so theability of Pif1 to concurrently bind multiple oligonucleotides wasinvestigated using Cy3- and Cy5-labeled noncomplementary 8-mers becausethese should bind to Pif1 with a 1:1 stoichiometry when consideringonly the helicase binding site. Upon titration of a mixture of theCy3- and Cy5-labeled oligonucleotides with Pif1, FRET should be observedif multiple oligonucleotides bind to a single Pif1 molecule or toadjacent Pif1 molecules in a Pif1 oligomer (Figure 3a). Pif1 has been reported to dimerize in the presence ofDNA.18 If each Pif1 monomer in the dimerbinds a separate oligonucleotide, the FRET probes should be in theproximity. Alternatively, sites on the surface of Pif1 could interactwith additional DNA molecules to juxtapose the fluorophores. We findthat Pif1 is able to bind multiple oligonucleotides simultaneously,resulting in an increased FRET efficiency (Figure 3b), similar to that observed previously for hepatitis C virusNS349 and human Rad52.50 A similar increase in the FRET efficiency was observedwhen the concentration of DNA (2 nM) was the same as that used inthe annealing experiments (Figure S2 of the SupportingInformation). This activity provides a mechanism by which Pif1can increase the local concentration of two DNA strands, which shouldfacilitate annealing.


Yeast Pif1 accelerates annealing of complementary DNA strands.

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

Pif1 can bind multiple strands of DNA. (a) Schematic illustrationof fluorescence titration. Two noncomplementary 8-mer oligonucleotides(100 nM) were mixed and then titrated with Pif1. Fluorescence emissionwas measured at 668 nm with excitation at 550 nm. (b) Increase inCy5 fluorescence emission as a function of Pif1 concentration (blue).Data were fit to the Hill equation to obtain the concentration ofprotein at which half of the maximal FRET change is observed, 200nM Pif1, and the Hill coefficient of 2.1. Duplicate experiments producedsimilar results. No increase in fluorescence was observed in controlreactions in which Pif1 was added to only the Cy3-labeled oligonucleotide(red) or only the Cy5-labeled oligonucleotide (black).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Pif1 can bind multiple strands of DNA. (a) Schematic illustrationof fluorescence titration. Two noncomplementary 8-mer oligonucleotides(100 nM) were mixed and then titrated with Pif1. Fluorescence emissionwas measured at 668 nm with excitation at 550 nm. (b) Increase inCy5 fluorescence emission as a function of Pif1 concentration (blue).Data were fit to the Hill equation to obtain the concentration ofprotein at which half of the maximal FRET change is observed, 200nM Pif1, and the Hill coefficient of 2.1. Duplicate experiments producedsimilar results. No increase in fluorescence was observed in controlreactions in which Pif1 was added to only the Cy3-labeled oligonucleotide(red) or only the Cy5-labeled oligonucleotide (black).
Mentions: A FRET binding experiment was designed to investigate the abilityof Pif1 to simultaneously bind multiple DNA strands (Figure 3a). Like other superfamily 1 helicases, Pif1 sequestersapproximately eight nucleotides on the basis of a repeating patternof nucleotide protection of approximately eight nucleotides in thepresence of Pif1 in KMnO4 footprinting experiments (FigureS1 of the Supporting Information), so theability of Pif1 to concurrently bind multiple oligonucleotides wasinvestigated using Cy3- and Cy5-labeled noncomplementary 8-mers becausethese should bind to Pif1 with a 1:1 stoichiometry when consideringonly the helicase binding site. Upon titration of a mixture of theCy3- and Cy5-labeled oligonucleotides with Pif1, FRET should be observedif multiple oligonucleotides bind to a single Pif1 molecule or toadjacent Pif1 molecules in a Pif1 oligomer (Figure 3a). Pif1 has been reported to dimerize in the presence ofDNA.18 If each Pif1 monomer in the dimerbinds a separate oligonucleotide, the FRET probes should be in theproximity. Alternatively, sites on the surface of Pif1 could interactwith additional DNA molecules to juxtapose the fluorophores. We findthat Pif1 is able to bind multiple oligonucleotides simultaneously,resulting in an increased FRET efficiency (Figure 3b), similar to that observed previously for hepatitis C virusNS349 and human Rad52.50 A similar increase in the FRET efficiency was observedwhen the concentration of DNA (2 nM) was the same as that used inthe annealing experiments (Figure S2 of the SupportingInformation). This activity provides a mechanism by which Pif1can increase the local concentration of two DNA strands, which shouldfacilitate 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