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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.

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DNA-binding activity of RAD54B26–225. (A) Plasmid ssDNA (20 μM in nucleotides, lanes 1–5) or plasmid superhelical dsDNA (10 μM in nucleotides, lanes 6–10) was incubated with RAD54B26–225 at 37°C for 20 min. The concentrations of Rad54B26–225 used in the DNA-binding experiments were 2.0 μM (lanes 2 and 7), 4.0 μM (lanes 3 and 8), 8.0 μM (lanes 4 and 9) and 16 μM (lanes 5 and 10). The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. Nc and sc indicate nicked circular and superhelical dsDNA, respectively. (B) A 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) was incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 10 min and the reaction mixtures were fractionated on a 1% agarose gel. (C) Salt concentration titration for the RAD54B26–225–polyA complex. RAD54B26–225 (16 μM) was incubated with a 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) in the reaction mixture containing the indicated concentrations of KCl. The reaction mixtures were fractionated on a 1% agarose gel. (D) Various branched DNA substrates (0.2 μM in molecules) were incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 20 min, and the reaction mixtures were fractionated on a 5% polyacrylamide gel, which was stained with ethidium bromide.
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Figure 2: DNA-binding activity of RAD54B26–225. (A) Plasmid ssDNA (20 μM in nucleotides, lanes 1–5) or plasmid superhelical dsDNA (10 μM in nucleotides, lanes 6–10) was incubated with RAD54B26–225 at 37°C for 20 min. The concentrations of Rad54B26–225 used in the DNA-binding experiments were 2.0 μM (lanes 2 and 7), 4.0 μM (lanes 3 and 8), 8.0 μM (lanes 4 and 9) and 16 μM (lanes 5 and 10). The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. Nc and sc indicate nicked circular and superhelical dsDNA, respectively. (B) A 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) was incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 10 min and the reaction mixtures were fractionated on a 1% agarose gel. (C) Salt concentration titration for the RAD54B26–225–polyA complex. RAD54B26–225 (16 μM) was incubated with a 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) in the reaction mixture containing the indicated concentrations of KCl. The reaction mixtures were fractionated on a 1% agarose gel. (D) Various branched DNA substrates (0.2 μM in molecules) were incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 20 min, and the reaction mixtures were fractionated on a 5% polyacrylamide gel, which was stained with ethidium bromide.

Mentions: The conserved region of RAD54B (amino acid residues 321–785) contains the helicase motifs involved in DNA binding. As expected, the full-length RAD54B has both ssDNA- and dsDNA-binding activities (28). In contrast, the less conserved N-terminal domain of RAD54B has no known DNA-binding motifs, and it is not known whether this domain binds to DNA. Therefore, we first examined the DNA-binding activity of RAD54B26–225, using plasmid ssDNA and dsDNA substrates. As shown in Figure 2A, RAD54B26–225 bound to both plasmid ssDNA and dsDNA. To further characterize the DNA-binding activity of RAD54B26–225, oligonucleotide substrates were used. RAD54B26–225 bound to a polyA ssDNA oligonucleotide, a substrate that is free of secondary structures (Figure 2B). The binding was observed at higher salt concentrations (Figure 2C), suggesting that RAD54B26–225 interacts with ssDNA through specific interactions and not by nonspecific ionic interactions. RAD54B26–225 also interacted with a dsDNA oligonucleotide, as well as DNA oligonucleotides with branched structures (Figure 2D). The binding experiments were performed using the same concentrations of the DNA substrates (0.2 μM) and RAD54B (2, 4, 8 and 16 μM), to facilitate comparisons between the results with different DNA substrates. We found that RAD54B26–225 exhibited slightly higher affinity for dsDNA than ssDNA (compare the amount of uncomplexed DNA between Figure 2B, lane 5 and 2D, lane 5). This was also apparent from the higher affinity for dsDNA than for DNA substrates with shorter duplex regions, such as the splayed arm and the 3′-tailed or 5′-tailed duplexes (Figure 2D, lanes 1–20). We also found that among the branched DNA substrates we tested, RAD54B26–225 displayed the highest affinity for 5′-flapped DNA and 3′-PX junction (Figure 2D, lanes 26–35). These results suggested that RAD54B26–225 may specifically function on branched DNA molecules.Figure 2.


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)

DNA-binding activity of RAD54B26–225. (A) Plasmid ssDNA (20 μM in nucleotides, lanes 1–5) or plasmid superhelical dsDNA (10 μM in nucleotides, lanes 6–10) was incubated with RAD54B26–225 at 37°C for 20 min. The concentrations of Rad54B26–225 used in the DNA-binding experiments were 2.0 μM (lanes 2 and 7), 4.0 μM (lanes 3 and 8), 8.0 μM (lanes 4 and 9) and 16 μM (lanes 5 and 10). The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. Nc and sc indicate nicked circular and superhelical dsDNA, respectively. (B) A 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) was incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 10 min and the reaction mixtures were fractionated on a 1% agarose gel. (C) Salt concentration titration for the RAD54B26–225–polyA complex. RAD54B26–225 (16 μM) was incubated with a 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) in the reaction mixture containing the indicated concentrations of KCl. The reaction mixtures were fractionated on a 1% agarose gel. (D) Various branched DNA substrates (0.2 μM in molecules) were incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 20 min, and the reaction mixtures were fractionated on a 5% polyacrylamide gel, which was stained with ethidium bromide.
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Figure 2: DNA-binding activity of RAD54B26–225. (A) Plasmid ssDNA (20 μM in nucleotides, lanes 1–5) or plasmid superhelical dsDNA (10 μM in nucleotides, lanes 6–10) was incubated with RAD54B26–225 at 37°C for 20 min. The concentrations of Rad54B26–225 used in the DNA-binding experiments were 2.0 μM (lanes 2 and 7), 4.0 μM (lanes 3 and 8), 8.0 μM (lanes 4 and 9) and 16 μM (lanes 5 and 10). The reaction mixtures were fractionated on a 1% agarose gel, which was stained with ethidium bromide. Nc and sc indicate nicked circular and superhelical dsDNA, respectively. (B) A 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) was incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 10 min and the reaction mixtures were fractionated on a 1% agarose gel. (C) Salt concentration titration for the RAD54B26–225–polyA complex. RAD54B26–225 (16 μM) was incubated with a 32P labeled single-stranded oligonucleotide (polyA 44-mer, 0.2 μM in molecules) in the reaction mixture containing the indicated concentrations of KCl. The reaction mixtures were fractionated on a 1% agarose gel. (D) Various branched DNA substrates (0.2 μM in molecules) were incubated with RAD54B26–225 (2, 4, 8 or 16 μM) at 37°C for 20 min, and the reaction mixtures were fractionated on a 5% polyacrylamide gel, which was stained with ethidium bromide.
Mentions: The conserved region of RAD54B (amino acid residues 321–785) contains the helicase motifs involved in DNA binding. As expected, the full-length RAD54B has both ssDNA- and dsDNA-binding activities (28). In contrast, the less conserved N-terminal domain of RAD54B has no known DNA-binding motifs, and it is not known whether this domain binds to DNA. Therefore, we first examined the DNA-binding activity of RAD54B26–225, using plasmid ssDNA and dsDNA substrates. As shown in Figure 2A, RAD54B26–225 bound to both plasmid ssDNA and dsDNA. To further characterize the DNA-binding activity of RAD54B26–225, oligonucleotide substrates were used. RAD54B26–225 bound to a polyA ssDNA oligonucleotide, a substrate that is free of secondary structures (Figure 2B). The binding was observed at higher salt concentrations (Figure 2C), suggesting that RAD54B26–225 interacts with ssDNA through specific interactions and not by nonspecific ionic interactions. RAD54B26–225 also interacted with a dsDNA oligonucleotide, as well as DNA oligonucleotides with branched structures (Figure 2D). The binding experiments were performed using the same concentrations of the DNA substrates (0.2 μM) and RAD54B (2, 4, 8 and 16 μM), to facilitate comparisons between the results with different DNA substrates. We found that RAD54B26–225 exhibited slightly higher affinity for dsDNA than ssDNA (compare the amount of uncomplexed DNA between Figure 2B, lane 5 and 2D, lane 5). This was also apparent from the higher affinity for dsDNA than for DNA substrates with shorter duplex regions, such as the splayed arm and the 3′-tailed or 5′-tailed duplexes (Figure 2D, lanes 1–20). We also found that among the branched DNA substrates we tested, RAD54B26–225 displayed the highest affinity for 5′-flapped DNA and 3′-PX junction (Figure 2D, lanes 26–35). These results suggested that RAD54B26–225 may specifically function on branched DNA molecules.Figure 2.

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