Limits...
Biochemical analysis of the N-terminal domain of human RAD54B.

Sarai N, Kagawa W, Fujikawa N, Saito K, Hikiba J, Tanaka K, Miyagawa K, Kurumizaka H, Yokoyama S - Nucleic Acids Res. (2008)

Bottom Line: Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153-214 and 296-340, were found to directly bind to the N-terminal domain of RAD54B.Thus, RAD54B binding may affect the quaternary structure of DMC1.These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

ABSTRACT
The human RAD54B protein is a paralog of the RAD54 protein, which plays important roles in homologous recombination. RAD54B contains an N-terminal region outside the SWI2/SNF2 domain that shares less conservation with the corresponding region in RAD54. The biochemical roles of this region of RAD54B are not known, although the corresponding region in RAD54 is known to physically interact with RAD51. In the present study, we have biochemically characterized an N-terminal fragment of RAD54B, consisting of amino acid residues 26-225 (RAD54B(26-225)). This fragment formed a stable dimer in solution and bound to branched DNA structures. RAD54B(26-225) also interacted with DMC1 in both the presence and absence of DNA. Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153-214 and 296-340, were found to directly bind to the N-terminal domain of RAD54B. A structural alignment of DMC1 with the Methanococcus voltae RadA protein, a homolog of DMC1 in the helical filament form, indicated that these RAD54B-binding sites are located near the ATP-binding site at the monomer-monomer interface in the DMC1 helical filament. Thus, RAD54B binding may affect the quaternary structure of DMC1. These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination.

Show MeSH

Related in: MedlinePlus

Interaction between DMC1 and RAD54B26–225 on DNA. (A) DNA-binding activity of DMC1. Increasing amounts of DMC1 (10, 20, 40 and 80 μM in lanes 2–5 and lanes 7–10, respectively) were incubated with φX174 ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides). Lanes 1 and 6 indicate negative control experiments without protein. The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. (B) RAD54B26–225 forms ternary complexes with DMC1 and DNA. A constant amount of DMC1 (40 μM) was incubated with φX174 circular ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides) at 37°C for 20min, followed by an incubation with increasing amounts of RAD54B26–225 (0, 2.0, 4.0, 8.0 and 16 μM in lanes 2–6 and lanes 9–13, respectively) at 37°C for 20 min. In lanes 7 and 14, RAD54B26–225 (16 μM) was incubated with ssDNA and dsDNA, but not DMC1, respectively. Lanes 1 and 8 indicate negative control experiments without protein. (C) Electroelution analysis of the protein–DNA complex. The protein–DNA complex detected in Figure 4B lane 13 was electroeluted from the agarose gel, and was analyzed by 12% SDS–PAGE gel (lane3). Lane 5 is the negative control experiment performed without dsDNA. Lane 1 indicates the molecular mass markers. Lanes 2 and 3 are one-tenth of the input DMC1 and RAD54B26–225, respectively. Nc and sc indicate nicked circular and superhelical dsDNA, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553597&req=5

Figure 4: Interaction between DMC1 and RAD54B26–225 on DNA. (A) DNA-binding activity of DMC1. Increasing amounts of DMC1 (10, 20, 40 and 80 μM in lanes 2–5 and lanes 7–10, respectively) were incubated with φX174 ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides). Lanes 1 and 6 indicate negative control experiments without protein. The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. (B) RAD54B26–225 forms ternary complexes with DMC1 and DNA. A constant amount of DMC1 (40 μM) was incubated with φX174 circular ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides) at 37°C for 20min, followed by an incubation with increasing amounts of RAD54B26–225 (0, 2.0, 4.0, 8.0 and 16 μM in lanes 2–6 and lanes 9–13, respectively) at 37°C for 20 min. In lanes 7 and 14, RAD54B26–225 (16 μM) was incubated with ssDNA and dsDNA, but not DMC1, respectively. Lanes 1 and 8 indicate negative control experiments without protein. (C) Electroelution analysis of the protein–DNA complex. The protein–DNA complex detected in Figure 4B lane 13 was electroeluted from the agarose gel, and was analyzed by 12% SDS–PAGE gel (lane3). Lane 5 is the negative control experiment performed without dsDNA. Lane 1 indicates the molecular mass markers. Lanes 2 and 3 are one-tenth of the input DMC1 and RAD54B26–225, respectively. Nc and sc indicate nicked circular and superhelical dsDNA, respectively.

Mentions: We next addressed whether RAD54B26–225 can interact with the DMC1 protein bound to either ssDNA or dsDNA. To do this, DMC1–DNA complexes were initially formed, followed by the addition of RAD54B26–225 and the resulting complexes were examined by a gel shift assay. To minimize the chance of RAD54B26–225 binding to the DMC1-free regions of the DNA molecule, we determined the concentrations of DMC1 required to nearly saturate the DNA substrates (Figure 4A, lanes 4 and 9). These concentrations of DMC1 were incubated with ssDNA or dsDNA, followed by the addition of RAD54B26–225 to the reaction mixture. As shown in Figure 4B (lanes 3–6 and lanes 10–13), increasing concentrations of RAD54B26–225 resulted in the supershifting of the DMC1–DNA complexes in the agarose gel. The supershifted complexes migrated differently from the RAD54B26–225–DNA complex (Figure 4B, lanes 7 and 14). These results indicated that RAD54B26–225 can form ternary complexes with DMC1 and either ssDNA or dsDNA. In the experiments shown in Figure 4B (lanes 13 and 14), the migration distances of the DMC1–RAD54B26–225–dsDNA ternary complex and the RAD54B26–225–dsDNA complex were nearly the same. To exclude the possibility that DMC1 had dissociated from the DNA, leaving behind the RAD54B26–225–dsDNA complex, we performed an electroelution of the protein–DNA complex, to investigate whether it contained DMC1. As confirmed by SDS–PAGE, both DMC1 and RAD54B26–225 were detected (Figure 4C), indicating that the DMC1–RAD54B26–225–dsDNA ternary complex was actually formed.Figure 4.


Biochemical analysis of the N-terminal domain of human RAD54B.

Sarai N, Kagawa W, Fujikawa N, Saito K, Hikiba J, Tanaka K, Miyagawa K, Kurumizaka H, Yokoyama S - Nucleic Acids Res. (2008)

Interaction between DMC1 and RAD54B26–225 on DNA. (A) DNA-binding activity of DMC1. Increasing amounts of DMC1 (10, 20, 40 and 80 μM in lanes 2–5 and lanes 7–10, respectively) were incubated with φX174 ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides). Lanes 1 and 6 indicate negative control experiments without protein. The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. (B) RAD54B26–225 forms ternary complexes with DMC1 and DNA. A constant amount of DMC1 (40 μM) was incubated with φX174 circular ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides) at 37°C for 20min, followed by an incubation with increasing amounts of RAD54B26–225 (0, 2.0, 4.0, 8.0 and 16 μM in lanes 2–6 and lanes 9–13, respectively) at 37°C for 20 min. In lanes 7 and 14, RAD54B26–225 (16 μM) was incubated with ssDNA and dsDNA, but not DMC1, respectively. Lanes 1 and 8 indicate negative control experiments without protein. (C) Electroelution analysis of the protein–DNA complex. The protein–DNA complex detected in Figure 4B lane 13 was electroeluted from the agarose gel, and was analyzed by 12% SDS–PAGE gel (lane3). Lane 5 is the negative control experiment performed without dsDNA. Lane 1 indicates the molecular mass markers. Lanes 2 and 3 are one-tenth of the input DMC1 and RAD54B26–225, respectively. Nc and sc indicate nicked circular and superhelical dsDNA, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553597&req=5

Figure 4: Interaction between DMC1 and RAD54B26–225 on DNA. (A) DNA-binding activity of DMC1. Increasing amounts of DMC1 (10, 20, 40 and 80 μM in lanes 2–5 and lanes 7–10, respectively) were incubated with φX174 ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides). Lanes 1 and 6 indicate negative control experiments without protein. The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. (B) RAD54B26–225 forms ternary complexes with DMC1 and DNA. A constant amount of DMC1 (40 μM) was incubated with φX174 circular ssDNA (20 μM in nucleotides) or φX174 superhelical dsDNA (10 μM in nucleotides) at 37°C for 20min, followed by an incubation with increasing amounts of RAD54B26–225 (0, 2.0, 4.0, 8.0 and 16 μM in lanes 2–6 and lanes 9–13, respectively) at 37°C for 20 min. In lanes 7 and 14, RAD54B26–225 (16 μM) was incubated with ssDNA and dsDNA, but not DMC1, respectively. Lanes 1 and 8 indicate negative control experiments without protein. (C) Electroelution analysis of the protein–DNA complex. The protein–DNA complex detected in Figure 4B lane 13 was electroeluted from the agarose gel, and was analyzed by 12% SDS–PAGE gel (lane3). Lane 5 is the negative control experiment performed without dsDNA. Lane 1 indicates the molecular mass markers. Lanes 2 and 3 are one-tenth of the input DMC1 and RAD54B26–225, respectively. Nc and sc indicate nicked circular and superhelical dsDNA, respectively.
Mentions: We next addressed whether RAD54B26–225 can interact with the DMC1 protein bound to either ssDNA or dsDNA. To do this, DMC1–DNA complexes were initially formed, followed by the addition of RAD54B26–225 and the resulting complexes were examined by a gel shift assay. To minimize the chance of RAD54B26–225 binding to the DMC1-free regions of the DNA molecule, we determined the concentrations of DMC1 required to nearly saturate the DNA substrates (Figure 4A, lanes 4 and 9). These concentrations of DMC1 were incubated with ssDNA or dsDNA, followed by the addition of RAD54B26–225 to the reaction mixture. As shown in Figure 4B (lanes 3–6 and lanes 10–13), increasing concentrations of RAD54B26–225 resulted in the supershifting of the DMC1–DNA complexes in the agarose gel. The supershifted complexes migrated differently from the RAD54B26–225–DNA complex (Figure 4B, lanes 7 and 14). These results indicated that RAD54B26–225 can form ternary complexes with DMC1 and either ssDNA or dsDNA. In the experiments shown in Figure 4B (lanes 13 and 14), the migration distances of the DMC1–RAD54B26–225–dsDNA ternary complex and the RAD54B26–225–dsDNA complex were nearly the same. To exclude the possibility that DMC1 had dissociated from the DNA, leaving behind the RAD54B26–225–dsDNA complex, we performed an electroelution of the protein–DNA complex, to investigate whether it contained DMC1. As confirmed by SDS–PAGE, both DMC1 and RAD54B26–225 were detected (Figure 4C), indicating that the DMC1–RAD54B26–225–dsDNA ternary complex was actually formed.Figure 4.

Bottom Line: Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153-214 and 296-340, were found to directly bind to the N-terminal domain of RAD54B.Thus, RAD54B binding may affect the quaternary structure of DMC1.These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

ABSTRACT
The human RAD54B protein is a paralog of the RAD54 protein, which plays important roles in homologous recombination. RAD54B contains an N-terminal region outside the SWI2/SNF2 domain that shares less conservation with the corresponding region in RAD54. The biochemical roles of this region of RAD54B are not known, although the corresponding region in RAD54 is known to physically interact with RAD51. In the present study, we have biochemically characterized an N-terminal fragment of RAD54B, consisting of amino acid residues 26-225 (RAD54B(26-225)). This fragment formed a stable dimer in solution and bound to branched DNA structures. RAD54B(26-225) also interacted with DMC1 in both the presence and absence of DNA. Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153-214 and 296-340, were found to directly bind to the N-terminal domain of RAD54B. A structural alignment of DMC1 with the Methanococcus voltae RadA protein, a homolog of DMC1 in the helical filament form, indicated that these RAD54B-binding sites are located near the ATP-binding site at the monomer-monomer interface in the DMC1 helical filament. Thus, RAD54B binding may affect the quaternary structure of DMC1. These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination.

Show MeSH
Related in: MedlinePlus