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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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Related in: MedlinePlus

Pif1-catalyzed annealingof partial duplex substrates with 3′-overhangs.(a) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and complementary 30nt overhangsto generate a 90bp blunt end dsDNA product. (b) Shown are resultsfor Pif1 (200 nM) annealing of 30nt-30bp (2.6 nM) and radiolabeled30bp-30nt (2 nM) to generate a 90bp blunt end DNA product in the presenceof ATP and MgCl2 (circles). Pif1 annealing in the presenceof 200 nM Rim1 (squares) and 200 nM RPA (diamonds) was also measured.Annealing activity of RPA alone is shown as empty circles, Rim1 aloneis shown as X. Spontaneous annealing in the absence of enzyme wasalso measured (triangles). Data were fit to a simple annealing mechanism(Scheme 2) to obtain second-order rate constantsfor annealing by Pif1 (9.2 × 106 M–1 s–1), Pif1 and RPA (1.5 × 107 M–1 s–1), Pif1 and Rim1 (4.8 ×106 M–1 s–1), and RPA(1.2 × 107 M–1 s–1). (c) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and 30nt overhangs containing 4ntof mismatch in the middle of otherwise complementary sequences togenerate a 90bp blunt end dsDNA product containing a 4nt bubble. (d)Results for Pif1 (200 nM)-catalyzed annealing of 30nt-30bp-mut (2.6nM) and radiolabeled 30bp-30nt (2 nM) to generate a 90bp blunt endDNA product containing a 4nt bubble in the presence of ATP and MgCl2 (circles). Spontaneous annealing in the absence of enzymewas also measured (triangles). Data were fit to a simple annealingmechanism (Scheme 2) to obtain a second-orderrate constant for annealing by Pif1 (4.1 × 106 M–1 s–1).
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fig7: Pif1-catalyzed annealingof partial duplex substrates with 3′-overhangs.(a) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and complementary 30nt overhangsto generate a 90bp blunt end dsDNA product. (b) Shown are resultsfor Pif1 (200 nM) annealing of 30nt-30bp (2.6 nM) and radiolabeled30bp-30nt (2 nM) to generate a 90bp blunt end DNA product in the presenceof ATP and MgCl2 (circles). Pif1 annealing in the presenceof 200 nM Rim1 (squares) and 200 nM RPA (diamonds) was also measured.Annealing activity of RPA alone is shown as empty circles, Rim1 aloneis shown as X. Spontaneous annealing in the absence of enzyme wasalso measured (triangles). Data were fit to a simple annealing mechanism(Scheme 2) to obtain second-order rate constantsfor annealing by Pif1 (9.2 × 106 M–1 s–1), Pif1 and RPA (1.5 × 107 M–1 s–1), Pif1 and Rim1 (4.8 ×106 M–1 s–1), and RPA(1.2 × 107 M–1 s–1). (c) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and 30nt overhangs containing 4ntof mismatch in the middle of otherwise complementary sequences togenerate a 90bp blunt end dsDNA product containing a 4nt bubble. (d)Results for Pif1 (200 nM)-catalyzed annealing of 30nt-30bp-mut (2.6nM) and radiolabeled 30bp-30nt (2 nM) to generate a 90bp blunt endDNA product containing a 4nt bubble in the presence of ATP and MgCl2 (circles). Spontaneous annealing in the absence of enzymewas also measured (triangles). Data were fit to a simple annealingmechanism (Scheme 2) to obtain a second-orderrate constant for annealing by Pif1 (4.1 × 106 M–1 s–1).

Mentions: Repair of double-strand breaks in cells can occur by various pathways,most of which involve resection by a nuclease, resulting in complementary3′-overhangs. Two partial duplex substrates (each containing30bp and a 30nt 3′-ssDNA overhang) (Figure 7a) were used to investigate the ability of Pif1 to catalyzeannealing of such substrates. These substrates are similar to thosethat could occur during double-strand break repair by the aNHEJ orMMEJ pathway, although sequences involved in aNHEJ often contain regionsof noncomplementarity within the region to be annealed. In the absenceof enzyme, very little annealing was observed; however, in the presenceof Pif1, annealing was much more rapid (Figure 7b). During double-strand break repair, after end resection, the resultingssDNA is thought to be coated by single-stranded DNA binding proteins.Because Pif1 is involved in genome maintenance in both the yeast nucleusand mitochondria, the ability of Pif1 to anneal in the presence ofyeast RPA, the nuclear single-stranded DNA binding protein, and Rim1,the yeast mitochondrial single-stranded DNA binding protein, was investigated(Figure 7b). Annealing was similar in the presenceand absence of single-stranded DNA binding proteins with rate constantsof 9.2 × 106, 1.5 × 107, and 4.8 ×106 M–1 s–1 for annealingby Pif1 alone, Pif1 and RPA, and Pif1 in the presence of Rim1, respectively.No annealing was observed by Rim1, but RPA alone annealed like Pif1alone with a rate constant of 1.2 × 107 M–1 s–1. Spontaneous annealing was negligible withinthis time frame. The ability of Pif1 to anneal substrates with 3′-overhangsin the presence of single-stranded DNA binding proteins suggests thatPif1 annealing activity could function in processes such as double-strandbreak repair in addition to its known role in the regulation of telomeraseat DSBs39−42 and its recent description in BIR.24−26


Yeast Pif1 accelerates annealing of complementary DNA strands.

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

Pif1-catalyzed annealingof partial duplex substrates with 3′-overhangs.(a) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and complementary 30nt overhangsto generate a 90bp blunt end dsDNA product. (b) Shown are resultsfor Pif1 (200 nM) annealing of 30nt-30bp (2.6 nM) and radiolabeled30bp-30nt (2 nM) to generate a 90bp blunt end DNA product in the presenceof ATP and MgCl2 (circles). Pif1 annealing in the presenceof 200 nM Rim1 (squares) and 200 nM RPA (diamonds) was also measured.Annealing activity of RPA alone is shown as empty circles, Rim1 aloneis shown as X. Spontaneous annealing in the absence of enzyme wasalso measured (triangles). Data were fit to a simple annealing mechanism(Scheme 2) to obtain second-order rate constantsfor annealing by Pif1 (9.2 × 106 M–1 s–1), Pif1 and RPA (1.5 × 107 M–1 s–1), Pif1 and Rim1 (4.8 ×106 M–1 s–1), and RPA(1.2 × 107 M–1 s–1). (c) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and 30nt overhangs containing 4ntof mismatch in the middle of otherwise complementary sequences togenerate a 90bp blunt end dsDNA product containing a 4nt bubble. (d)Results for Pif1 (200 nM)-catalyzed annealing of 30nt-30bp-mut (2.6nM) and radiolabeled 30bp-30nt (2 nM) to generate a 90bp blunt endDNA product containing a 4nt bubble in the presence of ATP and MgCl2 (circles). Spontaneous annealing in the absence of enzymewas also measured (triangles). Data were fit to a simple annealingmechanism (Scheme 2) to obtain a second-orderrate constant for annealing by Pif1 (4.1 × 106 M–1 s–1).
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fig7: Pif1-catalyzed annealingof partial duplex substrates with 3′-overhangs.(a) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and complementary 30nt overhangsto generate a 90bp blunt end dsDNA product. (b) Shown are resultsfor Pif1 (200 nM) annealing of 30nt-30bp (2.6 nM) and radiolabeled30bp-30nt (2 nM) to generate a 90bp blunt end DNA product in the presenceof ATP and MgCl2 (circles). Pif1 annealing in the presenceof 200 nM Rim1 (squares) and 200 nM RPA (diamonds) was also measured.Annealing activity of RPA alone is shown as empty circles, Rim1 aloneis shown as X. Spontaneous annealing in the absence of enzyme wasalso measured (triangles). Data were fit to a simple annealing mechanism(Scheme 2) to obtain second-order rate constantsfor annealing by Pif1 (9.2 × 106 M–1 s–1), Pif1 and RPA (1.5 × 107 M–1 s–1), Pif1 and Rim1 (4.8 ×106 M–1 s–1), and RPA(1.2 × 107 M–1 s–1). (c) Schematic illustration for strand annealing experiments usingtwo substrates with 30bp duplexes and 30nt overhangs containing 4ntof mismatch in the middle of otherwise complementary sequences togenerate a 90bp blunt end dsDNA product containing a 4nt bubble. (d)Results for Pif1 (200 nM)-catalyzed annealing of 30nt-30bp-mut (2.6nM) and radiolabeled 30bp-30nt (2 nM) to generate a 90bp blunt endDNA product containing a 4nt bubble in the presence of ATP and MgCl2 (circles). Spontaneous annealing in the absence of enzymewas also measured (triangles). Data were fit to a simple annealingmechanism (Scheme 2) to obtain a second-orderrate constant for annealing by Pif1 (4.1 × 106 M–1 s–1).
Mentions: Repair of double-strand breaks in cells can occur by various pathways,most of which involve resection by a nuclease, resulting in complementary3′-overhangs. Two partial duplex substrates (each containing30bp and a 30nt 3′-ssDNA overhang) (Figure 7a) were used to investigate the ability of Pif1 to catalyzeannealing of such substrates. These substrates are similar to thosethat could occur during double-strand break repair by the aNHEJ orMMEJ pathway, although sequences involved in aNHEJ often contain regionsof noncomplementarity within the region to be annealed. In the absenceof enzyme, very little annealing was observed; however, in the presenceof Pif1, annealing was much more rapid (Figure 7b). During double-strand break repair, after end resection, the resultingssDNA is thought to be coated by single-stranded DNA binding proteins.Because Pif1 is involved in genome maintenance in both the yeast nucleusand mitochondria, the ability of Pif1 to anneal in the presence ofyeast RPA, the nuclear single-stranded DNA binding protein, and Rim1,the yeast mitochondrial single-stranded DNA binding protein, was investigated(Figure 7b). Annealing was similar in the presenceand absence of single-stranded DNA binding proteins with rate constantsof 9.2 × 106, 1.5 × 107, and 4.8 ×106 M–1 s–1 for annealingby Pif1 alone, Pif1 and RPA, and Pif1 in the presence of Rim1, respectively.No annealing was observed by Rim1, but RPA alone annealed like Pif1alone with a rate constant of 1.2 × 107 M–1 s–1. Spontaneous annealing was negligible withinthis time frame. The ability of Pif1 to anneal substrates with 3′-overhangsin the presence of single-stranded DNA binding proteins suggests thatPif1 annealing activity could function in processes such as double-strandbreak repair in addition to its known role in the regulation of telomeraseat DSBs39−42 and its recent description in BIR.24−26

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