Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1 - PubMed (original) (raw)
Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1
G Petukhova et al. Genes Dev. 2000.
Abstract
The first DNA joint formed in homologous recombination processes is a D-loop. Saccharomyces cerevisiae RDH54/TID1-encoded product, a Swi2/Snf2-like factor involved in recombination, is shown here to promote D-loop formation with Rad51 recombinase. Physical interaction between Rdh54 and Rad51 is functionally important because Rdh54 does not enhance the recombinase activity of the Escherichia coli RecA protein. Robust dsDNA-activated ATPase activity in Rdh54 generates unconstrained negative and positive supercoils in DNA. Efficient D-loop formation occurs with even topologically relaxed DNA, suggesting that via specific protein-protein interactions, the negative supercoils produced by Rdh54 are used by Rad51 for making DNA joints.
Figures
Figure 1
Purification and DNA-dependent ATPase activity of Rdh54 and rdh54 K351R. (A) Immunoblot analysis. Nitrocellulose blot containing extracts from strain BJ5464 harboring the empty vector pPM231 (2μ, GAL–PGK; lane 1) and from BJ5464 harboring plasmid pRdh54.1 (2μ, GAL–PGK–6His–RDH54; lane 2) and plasmid prdh54K/R.1 (2μ, GAL–PGK–6His–rdh54 K351R; lane 3), along with 50 ng of purified Rdh54 (lane 4) and rdh54 K351R (lane 5), were subjected to immunoblot analysis with anti-Rdh54 antibodies. The arrow marks the position of the Rdh54 and rdh54 K351R proteins. The level of Rdh54 in wild-type extract (lane 1) is too low to detect under the conditions used. (B) An 8% SDS–polyacrylamide gel containing 2 μg of purified Rdh54 (lane 2) and rdh54 K351R (lane 3) was stained with Coomassie Blue. (C) Purified Rdh54 (40 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP in the absence of DNA (□) and in the presence of either φX viral (+) strand (▵; 30 μ
m
nucleotides) or φX replicative form I DNA (○; 15 μ
m
base pairs) for the indicated times at 37°C. The rdh54 K351R mutant protein (120 n
m
) was also incubated with φX replicative form I DNA (●; 15 μ
m
base pairs); no ATP hydrolysis was detected with rdh54K351R when DNA was omitted (data not shown). (D) Double-stranded DNA is the preferred cofactor for ATP hydrolysis. Rdh54 (30 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP with increasing concentrations of oligo 1 (□; in nucleotides), oligo 2 (▵; in nucleotides), or the duplex obtained by hybridizing oligo 1 and oligo 2 (○; in base pairs) for 8 min at 37°C.
Figure 1
Purification and DNA-dependent ATPase activity of Rdh54 and rdh54 K351R. (A) Immunoblot analysis. Nitrocellulose blot containing extracts from strain BJ5464 harboring the empty vector pPM231 (2μ, GAL–PGK; lane 1) and from BJ5464 harboring plasmid pRdh54.1 (2μ, GAL–PGK–6His–RDH54; lane 2) and plasmid prdh54K/R.1 (2μ, GAL–PGK–6His–rdh54 K351R; lane 3), along with 50 ng of purified Rdh54 (lane 4) and rdh54 K351R (lane 5), were subjected to immunoblot analysis with anti-Rdh54 antibodies. The arrow marks the position of the Rdh54 and rdh54 K351R proteins. The level of Rdh54 in wild-type extract (lane 1) is too low to detect under the conditions used. (B) An 8% SDS–polyacrylamide gel containing 2 μg of purified Rdh54 (lane 2) and rdh54 K351R (lane 3) was stained with Coomassie Blue. (C) Purified Rdh54 (40 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP in the absence of DNA (□) and in the presence of either φX viral (+) strand (▵; 30 μ
m
nucleotides) or φX replicative form I DNA (○; 15 μ
m
base pairs) for the indicated times at 37°C. The rdh54 K351R mutant protein (120 n
m
) was also incubated with φX replicative form I DNA (●; 15 μ
m
base pairs); no ATP hydrolysis was detected with rdh54K351R when DNA was omitted (data not shown). (D) Double-stranded DNA is the preferred cofactor for ATP hydrolysis. Rdh54 (30 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP with increasing concentrations of oligo 1 (□; in nucleotides), oligo 2 (▵; in nucleotides), or the duplex obtained by hybridizing oligo 1 and oligo 2 (○; in base pairs) for 8 min at 37°C.
Figure 1
Purification and DNA-dependent ATPase activity of Rdh54 and rdh54 K351R. (A) Immunoblot analysis. Nitrocellulose blot containing extracts from strain BJ5464 harboring the empty vector pPM231 (2μ, GAL–PGK; lane 1) and from BJ5464 harboring plasmid pRdh54.1 (2μ, GAL–PGK–6His–RDH54; lane 2) and plasmid prdh54K/R.1 (2μ, GAL–PGK–6His–rdh54 K351R; lane 3), along with 50 ng of purified Rdh54 (lane 4) and rdh54 K351R (lane 5), were subjected to immunoblot analysis with anti-Rdh54 antibodies. The arrow marks the position of the Rdh54 and rdh54 K351R proteins. The level of Rdh54 in wild-type extract (lane 1) is too low to detect under the conditions used. (B) An 8% SDS–polyacrylamide gel containing 2 μg of purified Rdh54 (lane 2) and rdh54 K351R (lane 3) was stained with Coomassie Blue. (C) Purified Rdh54 (40 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP in the absence of DNA (□) and in the presence of either φX viral (+) strand (▵; 30 μ
m
nucleotides) or φX replicative form I DNA (○; 15 μ
m
base pairs) for the indicated times at 37°C. The rdh54 K351R mutant protein (120 n
m
) was also incubated with φX replicative form I DNA (●; 15 μ
m
base pairs); no ATP hydrolysis was detected with rdh54K351R when DNA was omitted (data not shown). (D) Double-stranded DNA is the preferred cofactor for ATP hydrolysis. Rdh54 (30 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP with increasing concentrations of oligo 1 (□; in nucleotides), oligo 2 (▵; in nucleotides), or the duplex obtained by hybridizing oligo 1 and oligo 2 (○; in base pairs) for 8 min at 37°C.
Figure 1
Purification and DNA-dependent ATPase activity of Rdh54 and rdh54 K351R. (A) Immunoblot analysis. Nitrocellulose blot containing extracts from strain BJ5464 harboring the empty vector pPM231 (2μ, GAL–PGK; lane 1) and from BJ5464 harboring plasmid pRdh54.1 (2μ, GAL–PGK–6His–RDH54; lane 2) and plasmid prdh54K/R.1 (2μ, GAL–PGK–6His–rdh54 K351R; lane 3), along with 50 ng of purified Rdh54 (lane 4) and rdh54 K351R (lane 5), were subjected to immunoblot analysis with anti-Rdh54 antibodies. The arrow marks the position of the Rdh54 and rdh54 K351R proteins. The level of Rdh54 in wild-type extract (lane 1) is too low to detect under the conditions used. (B) An 8% SDS–polyacrylamide gel containing 2 μg of purified Rdh54 (lane 2) and rdh54 K351R (lane 3) was stained with Coomassie Blue. (C) Purified Rdh54 (40 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP in the absence of DNA (□) and in the presence of either φX viral (+) strand (▵; 30 μ
m
nucleotides) or φX replicative form I DNA (○; 15 μ
m
base pairs) for the indicated times at 37°C. The rdh54 K351R mutant protein (120 n
m
) was also incubated with φX replicative form I DNA (●; 15 μ
m
base pairs); no ATP hydrolysis was detected with rdh54K351R when DNA was omitted (data not shown). (D) Double-stranded DNA is the preferred cofactor for ATP hydrolysis. Rdh54 (30 n
m
) was incubated with 1.5 m
m
[γ-32P]ATP with increasing concentrations of oligo 1 (□; in nucleotides), oligo 2 (▵; in nucleotides), or the duplex obtained by hybridizing oligo 1 and oligo 2 (○; in base pairs) for 8 min at 37°C.
Figure 2
Rdh54 interacts with Rad51 and promotes D-loop formation. (A)Rad51 and Rdh54 interact physically. Rdh54 (panel I) or rdh54 K351R (panel II) was incubated with Affi-Gel 15 beads bearing bovine serum albumin (Affi-BSA) or Rad51 (Affi-Rad51). The beads were then washed with 100 m
m
KCl and eluted with 3% SDS. The input material (I), the supernatant containing unbound Rdh54 or rdh54 K351R (S), the KCl wash (W), and the SDS eluate (E) were run in an 8% polyacrylamide gel and stained with Coomassie Blue. Densitometric scanning of the gels revealed that > 80% of Rdh54 was retained on the Affi-Rad51 beads, whereas the Affi-BSA control beads bound < 3% of the Rdh54 protein. (B) Schematic of the D-loop reaction. Linear viral (+) strand (designated ss) is paired with the replicative form I DNA (designated sc) to yield a D-loop. (C) ATP hydrolysis-dependent promotion of D-loop formation by Rdh54. Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with φX replicative form I DNA (sc) at 23°C for the indicated times (lanes 2–9). The reaction mixture in lane 8 was heated at 92°C for 2 min before electrophoresis; ATP was omitted from the reaction mixture in lane 9. In lanes 10–12, Rad51, rdh54 K351R (rdh K/R), and RPA were incubated with the DNA substrates at 23°C for the indicated times. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). In addition to the main D-loop species, more complex D-loop species are seen in the reaction involving wild-type Rdh54, especially in the later time points (lanes 2–7). (D) The gel in C was subjected to image analysis, and the data points were plotted. (●) results in lanes 2–7 of C involving wild-type Rdh54; (○) results in lanes 10–12 of C involving rdh54 K351R. (E) Requirements for D-loop formation. Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated in buffer without any recombination protein (lane 1), with Rad51, RPA, and Rdh54 (lane 2), with RPA and Rdh54 (lane 3), with Rad51 and RPA (lane 4), with Rad51 and Rdh54 (lane 5), and with Rad51, E. coli SSB, and Rdh54 (lane 6). In lane 7, φX replicative form I alone, and in lane 8, linear φX (+) strand alone, was incubated with Rad51, RPA, and Rdh54. In lanes 9 and 10, linear φX (+) strand and the unrelated pBlueScript replicative form I DNA (sc, pBS) were incubated in buffer without any recombination protein (lane 9) or with the combination of Rad51, RPA, and Rdh54 (lane 10). The reaction temperature was 23°C.
Figure 2
Rdh54 interacts with Rad51 and promotes D-loop formation. (A)Rad51 and Rdh54 interact physically. Rdh54 (panel I) or rdh54 K351R (panel II) was incubated with Affi-Gel 15 beads bearing bovine serum albumin (Affi-BSA) or Rad51 (Affi-Rad51). The beads were then washed with 100 m
m
KCl and eluted with 3% SDS. The input material (I), the supernatant containing unbound Rdh54 or rdh54 K351R (S), the KCl wash (W), and the SDS eluate (E) were run in an 8% polyacrylamide gel and stained with Coomassie Blue. Densitometric scanning of the gels revealed that > 80% of Rdh54 was retained on the Affi-Rad51 beads, whereas the Affi-BSA control beads bound < 3% of the Rdh54 protein. (B) Schematic of the D-loop reaction. Linear viral (+) strand (designated ss) is paired with the replicative form I DNA (designated sc) to yield a D-loop. (C) ATP hydrolysis-dependent promotion of D-loop formation by Rdh54. Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with φX replicative form I DNA (sc) at 23°C for the indicated times (lanes 2–9). The reaction mixture in lane 8 was heated at 92°C for 2 min before electrophoresis; ATP was omitted from the reaction mixture in lane 9. In lanes 10–12, Rad51, rdh54 K351R (rdh K/R), and RPA were incubated with the DNA substrates at 23°C for the indicated times. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). In addition to the main D-loop species, more complex D-loop species are seen in the reaction involving wild-type Rdh54, especially in the later time points (lanes 2–7). (D) The gel in C was subjected to image analysis, and the data points were plotted. (●) results in lanes 2–7 of C involving wild-type Rdh54; (○) results in lanes 10–12 of C involving rdh54 K351R. (E) Requirements for D-loop formation. Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated in buffer without any recombination protein (lane 1), with Rad51, RPA, and Rdh54 (lane 2), with RPA and Rdh54 (lane 3), with Rad51 and RPA (lane 4), with Rad51 and Rdh54 (lane 5), and with Rad51, E. coli SSB, and Rdh54 (lane 6). In lane 7, φX replicative form I alone, and in lane 8, linear φX (+) strand alone, was incubated with Rad51, RPA, and Rdh54. In lanes 9 and 10, linear φX (+) strand and the unrelated pBlueScript replicative form I DNA (sc, pBS) were incubated in buffer without any recombination protein (lane 9) or with the combination of Rad51, RPA, and Rdh54 (lane 10). The reaction temperature was 23°C.
Figure 2
Rdh54 interacts with Rad51 and promotes D-loop formation. (A)Rad51 and Rdh54 interact physically. Rdh54 (panel I) or rdh54 K351R (panel II) was incubated with Affi-Gel 15 beads bearing bovine serum albumin (Affi-BSA) or Rad51 (Affi-Rad51). The beads were then washed with 100 m
m
KCl and eluted with 3% SDS. The input material (I), the supernatant containing unbound Rdh54 or rdh54 K351R (S), the KCl wash (W), and the SDS eluate (E) were run in an 8% polyacrylamide gel and stained with Coomassie Blue. Densitometric scanning of the gels revealed that > 80% of Rdh54 was retained on the Affi-Rad51 beads, whereas the Affi-BSA control beads bound < 3% of the Rdh54 protein. (B) Schematic of the D-loop reaction. Linear viral (+) strand (designated ss) is paired with the replicative form I DNA (designated sc) to yield a D-loop. (C) ATP hydrolysis-dependent promotion of D-loop formation by Rdh54. Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with φX replicative form I DNA (sc) at 23°C for the indicated times (lanes 2–9). The reaction mixture in lane 8 was heated at 92°C for 2 min before electrophoresis; ATP was omitted from the reaction mixture in lane 9. In lanes 10–12, Rad51, rdh54 K351R (rdh K/R), and RPA were incubated with the DNA substrates at 23°C for the indicated times. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). In addition to the main D-loop species, more complex D-loop species are seen in the reaction involving wild-type Rdh54, especially in the later time points (lanes 2–7). (D) The gel in C was subjected to image analysis, and the data points were plotted. (●) results in lanes 2–7 of C involving wild-type Rdh54; (○) results in lanes 10–12 of C involving rdh54 K351R. (E) Requirements for D-loop formation. Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated in buffer without any recombination protein (lane 1), with Rad51, RPA, and Rdh54 (lane 2), with RPA and Rdh54 (lane 3), with Rad51 and RPA (lane 4), with Rad51 and Rdh54 (lane 5), and with Rad51, E. coli SSB, and Rdh54 (lane 6). In lane 7, φX replicative form I alone, and in lane 8, linear φX (+) strand alone, was incubated with Rad51, RPA, and Rdh54. In lanes 9 and 10, linear φX (+) strand and the unrelated pBlueScript replicative form I DNA (sc, pBS) were incubated in buffer without any recombination protein (lane 9) or with the combination of Rad51, RPA, and Rdh54 (lane 10). The reaction temperature was 23°C.
Figure 2
Rdh54 interacts with Rad51 and promotes D-loop formation. (A)Rad51 and Rdh54 interact physically. Rdh54 (panel I) or rdh54 K351R (panel II) was incubated with Affi-Gel 15 beads bearing bovine serum albumin (Affi-BSA) or Rad51 (Affi-Rad51). The beads were then washed with 100 m
m
KCl and eluted with 3% SDS. The input material (I), the supernatant containing unbound Rdh54 or rdh54 K351R (S), the KCl wash (W), and the SDS eluate (E) were run in an 8% polyacrylamide gel and stained with Coomassie Blue. Densitometric scanning of the gels revealed that > 80% of Rdh54 was retained on the Affi-Rad51 beads, whereas the Affi-BSA control beads bound < 3% of the Rdh54 protein. (B) Schematic of the D-loop reaction. Linear viral (+) strand (designated ss) is paired with the replicative form I DNA (designated sc) to yield a D-loop. (C) ATP hydrolysis-dependent promotion of D-loop formation by Rdh54. Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with φX replicative form I DNA (sc) at 23°C for the indicated times (lanes 2–9). The reaction mixture in lane 8 was heated at 92°C for 2 min before electrophoresis; ATP was omitted from the reaction mixture in lane 9. In lanes 10–12, Rad51, rdh54 K351R (rdh K/R), and RPA were incubated with the DNA substrates at 23°C for the indicated times. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). In addition to the main D-loop species, more complex D-loop species are seen in the reaction involving wild-type Rdh54, especially in the later time points (lanes 2–7). (D) The gel in C was subjected to image analysis, and the data points were plotted. (●) results in lanes 2–7 of C involving wild-type Rdh54; (○) results in lanes 10–12 of C involving rdh54 K351R. (E) Requirements for D-loop formation. Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated in buffer without any recombination protein (lane 1), with Rad51, RPA, and Rdh54 (lane 2), with RPA and Rdh54 (lane 3), with Rad51 and RPA (lane 4), with Rad51 and Rdh54 (lane 5), and with Rad51, E. coli SSB, and Rdh54 (lane 6). In lane 7, φX replicative form I alone, and in lane 8, linear φX (+) strand alone, was incubated with Rad51, RPA, and Rdh54. In lanes 9 and 10, linear φX (+) strand and the unrelated pBlueScript replicative form I DNA (sc, pBS) were incubated in buffer without any recombination protein (lane 9) or with the combination of Rad51, RPA, and Rdh54 (lane 10). The reaction temperature was 23°C.
Figure 2
Rdh54 interacts with Rad51 and promotes D-loop formation. (A)Rad51 and Rdh54 interact physically. Rdh54 (panel I) or rdh54 K351R (panel II) was incubated with Affi-Gel 15 beads bearing bovine serum albumin (Affi-BSA) or Rad51 (Affi-Rad51). The beads were then washed with 100 m
m
KCl and eluted with 3% SDS. The input material (I), the supernatant containing unbound Rdh54 or rdh54 K351R (S), the KCl wash (W), and the SDS eluate (E) were run in an 8% polyacrylamide gel and stained with Coomassie Blue. Densitometric scanning of the gels revealed that > 80% of Rdh54 was retained on the Affi-Rad51 beads, whereas the Affi-BSA control beads bound < 3% of the Rdh54 protein. (B) Schematic of the D-loop reaction. Linear viral (+) strand (designated ss) is paired with the replicative form I DNA (designated sc) to yield a D-loop. (C) ATP hydrolysis-dependent promotion of D-loop formation by Rdh54. Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with φX replicative form I DNA (sc) at 23°C for the indicated times (lanes 2–9). The reaction mixture in lane 8 was heated at 92°C for 2 min before electrophoresis; ATP was omitted from the reaction mixture in lane 9. In lanes 10–12, Rad51, rdh54 K351R (rdh K/R), and RPA were incubated with the DNA substrates at 23°C for the indicated times. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). In addition to the main D-loop species, more complex D-loop species are seen in the reaction involving wild-type Rdh54, especially in the later time points (lanes 2–7). (D) The gel in C was subjected to image analysis, and the data points were plotted. (●) results in lanes 2–7 of C involving wild-type Rdh54; (○) results in lanes 10–12 of C involving rdh54 K351R. (E) Requirements for D-loop formation. Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated in buffer without any recombination protein (lane 1), with Rad51, RPA, and Rdh54 (lane 2), with RPA and Rdh54 (lane 3), with Rad51 and RPA (lane 4), with Rad51 and Rdh54 (lane 5), and with Rad51, E. coli SSB, and Rdh54 (lane 6). In lane 7, φX replicative form I alone, and in lane 8, linear φX (+) strand alone, was incubated with Rad51, RPA, and Rdh54. In lanes 9 and 10, linear φX (+) strand and the unrelated pBlueScript replicative form I DNA (sc, pBS) were incubated in buffer without any recombination protein (lane 9) or with the combination of Rad51, RPA, and Rdh54 (lane 10). The reaction temperature was 23°C.
Figure 3
DNA topology modification as revealed by treatment with E. coli topoisomerase I. (A) Increasing concentrations of Rdh54 (22, 44, 88, 220, and 440 n
m
in lanes 2–6, respectively) were incubated with relaxed φX DNA (18.5 μ
m
base pairs) in the presence of ATP and E. coli topoisomerase I; the DNA species were resolved in a 0.9% agarose gel and visualized by staining with ethidium bromide. In lane 1, the DNA was incubated in buffer with topoisomerase but no Rdh54; in lane 7, the DNA was incubated with 440 n
m
Rdh54 in the absence of topoisomerase. The product of DNA topology modification is designated Form OW. (B) ATP hydrolysis is required for DNA topology modification. In lanes 2–5, 200 n
m
Rdh54 was incubated with relaxed DNA and E. coli topoisomerase I in the absence of a nucleotide or in the presence of ATP, ATP-γ-S, or ADP as indicated, and the DNA product was analyzed as described in A. The rdh54 K351R mutant protein (200 and 400 n
m
in lanes 6 and 7, respectively) was also examined for the ability to remodel DNA. In lane 1, relaxed DNA was incubated in buffer with topoisomerase and ATP but no Rdh54 or rdh54 K351R. The DNA concentration was 18.5 μ
m
base pairs in all of the reaction mixtures. (C) Two-dimensional gel analysis of Form OW DNA. In panels I and II, negatively supercoiled φX DNA isolated from cells (ς = −0.06) without (I) or with (II) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. In panels III and IV, a mixture of negatively supercoiled φX DNA and purified Form OW DNA without (III) or with (IV) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. Note that the negatively supercoiled DNA, but not Form OW DNA, was relaxed by E. coli topoisomerase I (IV). In these gel analyses, the first dimension was conducted in the absence of chloroquine (−CQ) and the second dimension in the presence of chloroquine (+CQ). (nc) nicked circular DNA;(ow) Form OW DNA; (Rl) relaxed DNA; (sc) negatively supercoiled DNA.
Figure 3
DNA topology modification as revealed by treatment with E. coli topoisomerase I. (A) Increasing concentrations of Rdh54 (22, 44, 88, 220, and 440 n
m
in lanes 2–6, respectively) were incubated with relaxed φX DNA (18.5 μ
m
base pairs) in the presence of ATP and E. coli topoisomerase I; the DNA species were resolved in a 0.9% agarose gel and visualized by staining with ethidium bromide. In lane 1, the DNA was incubated in buffer with topoisomerase but no Rdh54; in lane 7, the DNA was incubated with 440 n
m
Rdh54 in the absence of topoisomerase. The product of DNA topology modification is designated Form OW. (B) ATP hydrolysis is required for DNA topology modification. In lanes 2–5, 200 n
m
Rdh54 was incubated with relaxed DNA and E. coli topoisomerase I in the absence of a nucleotide or in the presence of ATP, ATP-γ-S, or ADP as indicated, and the DNA product was analyzed as described in A. The rdh54 K351R mutant protein (200 and 400 n
m
in lanes 6 and 7, respectively) was also examined for the ability to remodel DNA. In lane 1, relaxed DNA was incubated in buffer with topoisomerase and ATP but no Rdh54 or rdh54 K351R. The DNA concentration was 18.5 μ
m
base pairs in all of the reaction mixtures. (C) Two-dimensional gel analysis of Form OW DNA. In panels I and II, negatively supercoiled φX DNA isolated from cells (ς = −0.06) without (I) or with (II) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. In panels III and IV, a mixture of negatively supercoiled φX DNA and purified Form OW DNA without (III) or with (IV) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. Note that the negatively supercoiled DNA, but not Form OW DNA, was relaxed by E. coli topoisomerase I (IV). In these gel analyses, the first dimension was conducted in the absence of chloroquine (−CQ) and the second dimension in the presence of chloroquine (+CQ). (nc) nicked circular DNA;(ow) Form OW DNA; (Rl) relaxed DNA; (sc) negatively supercoiled DNA.
Figure 3
DNA topology modification as revealed by treatment with E. coli topoisomerase I. (A) Increasing concentrations of Rdh54 (22, 44, 88, 220, and 440 n
m
in lanes 2–6, respectively) were incubated with relaxed φX DNA (18.5 μ
m
base pairs) in the presence of ATP and E. coli topoisomerase I; the DNA species were resolved in a 0.9% agarose gel and visualized by staining with ethidium bromide. In lane 1, the DNA was incubated in buffer with topoisomerase but no Rdh54; in lane 7, the DNA was incubated with 440 n
m
Rdh54 in the absence of topoisomerase. The product of DNA topology modification is designated Form OW. (B) ATP hydrolysis is required for DNA topology modification. In lanes 2–5, 200 n
m
Rdh54 was incubated with relaxed DNA and E. coli topoisomerase I in the absence of a nucleotide or in the presence of ATP, ATP-γ-S, or ADP as indicated, and the DNA product was analyzed as described in A. The rdh54 K351R mutant protein (200 and 400 n
m
in lanes 6 and 7, respectively) was also examined for the ability to remodel DNA. In lane 1, relaxed DNA was incubated in buffer with topoisomerase and ATP but no Rdh54 or rdh54 K351R. The DNA concentration was 18.5 μ
m
base pairs in all of the reaction mixtures. (C) Two-dimensional gel analysis of Form OW DNA. In panels I and II, negatively supercoiled φX DNA isolated from cells (ς = −0.06) without (I) or with (II) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. In panels III and IV, a mixture of negatively supercoiled φX DNA and purified Form OW DNA without (III) or with (IV) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. Note that the negatively supercoiled DNA, but not Form OW DNA, was relaxed by E. coli topoisomerase I (IV). In these gel analyses, the first dimension was conducted in the absence of chloroquine (−CQ) and the second dimension in the presence of chloroquine (+CQ). (nc) nicked circular DNA;(ow) Form OW DNA; (Rl) relaxed DNA; (sc) negatively supercoiled DNA.
Figure 4
DNA topology modification as revealed by treatment with calf thymus topoisomerase I. (A) Increasing concentrations of Rdh54 (0.088, 0.22, 0.44, 0.88, 1.32, and 1.76 μ
m
in lanes 2–7, respectively) were incubated with relaxed φX DNA (18.5 μ
m
base pairs) in the presence of 2 m
m
ATP and 4 units of calf thymus topoisomerase I; the DNA products were analyzed in a 0.9% agarose gel and visualized by staining with ethidium bromide. In lane 1, the DNA was incubated in buffer with topoisomerase but no Rdh54; in lane 8, the DNA was incubated with 1.76 μ
m
Rdh54 in the absence of topoisomerase. The product of DNA topology modification is designated Form UW. (B) ATP hydrolysis is required for DNA topology modification. Rdh54 (0.88 μ
m
in lanes 2–5) was incubated with relaxed DNA and calf thymus topoisomerase I in the absence of a nucleotide or in the presence of ATP, ATP-γ-S, or ADP as indicated; the DNA product was analyzed by electrophoresis as described in A. In lane 1, relaxed DNA was incubated in buffer with topoisomerase but no Rdh54. The rdh54 K351R mutant protein (1 and 2 μ
m
, in lanes 6 and 7, respectively) was also analyzed for the ability to remodel DNA. In lane 1, relaxed DNA was incubated in buffer with topoisomerase but no Rdh54 or rdh54 K351R. The DNA concentration was 18.5 μ
m
base pairs in all the reaction mixtures. (C) Two-dimensional gel analysis of Form UW DNA. In panel I, a mixture of relaxed φX DNA (Rl), slightly negatively supercoiled φX DNA (SC1; average ς = −0.017), and negatively supercoiled φX DNA isolated from cells (SC2; average ς = −0.06) was subjected to two-dimensional gel analysis in which the first dimension was conducted in the presence of chloroquine diphosphate (+CQ) and the second dimension was conducted without it (−CQ). Analysis of Form UW DNA made by treating relaxed φX DNA (18.5 μ
m
base pairs) with 0.6 μ
m
(panel II) and 0.8 μ
m
(panel III) Rdh54 and topoisomerase was also performed.
Figure 4
DNA topology modification as revealed by treatment with calf thymus topoisomerase I. (A) Increasing concentrations of Rdh54 (0.088, 0.22, 0.44, 0.88, 1.32, and 1.76 μ
m
in lanes 2–7, respectively) were incubated with relaxed φX DNA (18.5 μ
m
base pairs) in the presence of 2 m
m
ATP and 4 units of calf thymus topoisomerase I; the DNA products were analyzed in a 0.9% agarose gel and visualized by staining with ethidium bromide. In lane 1, the DNA was incubated in buffer with topoisomerase but no Rdh54; in lane 8, the DNA was incubated with 1.76 μ
m
Rdh54 in the absence of topoisomerase. The product of DNA topology modification is designated Form UW. (B) ATP hydrolysis is required for DNA topology modification. Rdh54 (0.88 μ
m
in lanes 2–5) was incubated with relaxed DNA and calf thymus topoisomerase I in the absence of a nucleotide or in the presence of ATP, ATP-γ-S, or ADP as indicated; the DNA product was analyzed by electrophoresis as described in A. In lane 1, relaxed DNA was incubated in buffer with topoisomerase but no Rdh54. The rdh54 K351R mutant protein (1 and 2 μ
m
, in lanes 6 and 7, respectively) was also analyzed for the ability to remodel DNA. In lane 1, relaxed DNA was incubated in buffer with topoisomerase but no Rdh54 or rdh54 K351R. The DNA concentration was 18.5 μ
m
base pairs in all the reaction mixtures. (C) Two-dimensional gel analysis of Form UW DNA. In panel I, a mixture of relaxed φX DNA (Rl), slightly negatively supercoiled φX DNA (SC1; average ς = −0.017), and negatively supercoiled φX DNA isolated from cells (SC2; average ς = −0.06) was subjected to two-dimensional gel analysis in which the first dimension was conducted in the presence of chloroquine diphosphate (+CQ) and the second dimension was conducted without it (−CQ). Analysis of Form UW DNA made by treating relaxed φX DNA (18.5 μ
m
base pairs) with 0.6 μ
m
(panel II) and 0.8 μ
m
(panel III) Rdh54 and topoisomerase was also performed.
Figure 4
DNA topology modification as revealed by treatment with calf thymus topoisomerase I. (A) Increasing concentrations of Rdh54 (0.088, 0.22, 0.44, 0.88, 1.32, and 1.76 μ
m
in lanes 2–7, respectively) were incubated with relaxed φX DNA (18.5 μ
m
base pairs) in the presence of 2 m
m
ATP and 4 units of calf thymus topoisomerase I; the DNA products were analyzed in a 0.9% agarose gel and visualized by staining with ethidium bromide. In lane 1, the DNA was incubated in buffer with topoisomerase but no Rdh54; in lane 8, the DNA was incubated with 1.76 μ
m
Rdh54 in the absence of topoisomerase. The product of DNA topology modification is designated Form UW. (B) ATP hydrolysis is required for DNA topology modification. Rdh54 (0.88 μ
m
in lanes 2–5) was incubated with relaxed DNA and calf thymus topoisomerase I in the absence of a nucleotide or in the presence of ATP, ATP-γ-S, or ADP as indicated; the DNA product was analyzed by electrophoresis as described in A. In lane 1, relaxed DNA was incubated in buffer with topoisomerase but no Rdh54. The rdh54 K351R mutant protein (1 and 2 μ
m
, in lanes 6 and 7, respectively) was also analyzed for the ability to remodel DNA. In lane 1, relaxed DNA was incubated in buffer with topoisomerase but no Rdh54 or rdh54 K351R. The DNA concentration was 18.5 μ
m
base pairs in all the reaction mixtures. (C) Two-dimensional gel analysis of Form UW DNA. In panel I, a mixture of relaxed φX DNA (Rl), slightly negatively supercoiled φX DNA (SC1; average ς = −0.017), and negatively supercoiled φX DNA isolated from cells (SC2; average ς = −0.06) was subjected to two-dimensional gel analysis in which the first dimension was conducted in the presence of chloroquine diphosphate (+CQ) and the second dimension was conducted without it (−CQ). Analysis of Form UW DNA made by treating relaxed φX DNA (18.5 μ
m
base pairs) with 0.6 μ
m
(panel II) and 0.8 μ
m
(panel III) Rdh54 and topoisomerase was also performed.
Figure 5
Dependence of D-loop formation on negative supercoiling. (A) Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with positively supercoiled DNA (+ sc;, panel I), topologically relaxed DNA (Rl; ς = 0; panel II), or negatively supercoiled DNA (sc) substrates with ς values of −0.021 (panel III) and −0.039 (panel IV) at 23°C for the indicated times. To effect separation of the D-loop from other DNA species, we analyzed the reaction mixtures in agarose gels containing 10 μ
m
ethidium bromide. (B) The data points from image analyses of the gels in A and from another reaction performed under the same conditions but with duplex substrate having a ς value of −0.076 are plotted.
Figure 5
Dependence of D-loop formation on negative supercoiling. (A) Linear φX viral (+) strand (ss) was incubated with Rad51, Rdh54, and RPA, and the nucleoprotein complex thus formed was reacted with positively supercoiled DNA (+ sc;, panel I), topologically relaxed DNA (Rl; ς = 0; panel II), or negatively supercoiled DNA (sc) substrates with ς values of −0.021 (panel III) and −0.039 (panel IV) at 23°C for the indicated times. To effect separation of the D-loop from other DNA species, we analyzed the reaction mixtures in agarose gels containing 10 μ
m
ethidium bromide. (B) The data points from image analyses of the gels in A and from another reaction performed under the same conditions but with duplex substrate having a ς value of −0.076 are plotted.
Figure 6
Effect of Rdh54 is specific for Rad51. (A)Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated with RecA and SSB (lanes 2–11) in the absence (RecA; lanes 2–6) or presence (RecA/Rdh; lanes 7–11) of Rdh54 for the times indicated. In lanes 12–14, the DNA substrates were incubated with Rad51, RPA, and Rdh54 (Rad51/Rdh) for the times indicated. The reaction temperature was 23°C, and the reaction mixtures were analyzed in an agarose gel without ethidium bromide. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). (B) The data points from image analysis of the gel in A are plotted. (●) results in lanes 2–6 of A involving RecA and SSB; (○) results in lanes 7–11 of A involving RecA, SSB, and Rdh54; (▵) results in lanes 12–14 of A involving Rad51, RPA, and Rdh54.
Figure 6
Effect of Rdh54 is specific for Rad51. (A)Linear φX viral (+) strand (ss) and replicative form I DNA (sc) were incubated with RecA and SSB (lanes 2–11) in the absence (RecA; lanes 2–6) or presence (RecA/Rdh; lanes 7–11) of Rdh54 for the times indicated. In lanes 12–14, the DNA substrates were incubated with Rad51, RPA, and Rdh54 (Rad51/Rdh) for the times indicated. The reaction temperature was 23°C, and the reaction mixtures were analyzed in an agarose gel without ethidium bromide. In lane 1, the DNA substrates were incubated in buffer without any recombination protein (Bl). (B) The data points from image analysis of the gel in A are plotted. (●) results in lanes 2–6 of A involving RecA and SSB; (○) results in lanes 7–11 of A involving RecA, SSB, and Rdh54; (▵) results in lanes 12–14 of A involving Rad51, RPA, and Rdh54.
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