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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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RAD54B26–225 interacts with RAD51 and DMC1. The interactions were observed by a pull-down assay, in which DMC1 (A) or RAD51 (B) was mixed with RAD54B26–225 that was covalently conjugated to an Affi-Gel 15 matrix. The proteins bound to the RAD54B26–225-conjugated beads were eluted by SDS–PAGE sample buffer, and fractionated on a 12% SDS–PAGE gel. Lanes 2 and 3 are one-tenth of the total proteins used. Lane 4 is the negative control using the Affi-Gel 15 matrix without RAD54B26–225. The salt concentration was titrated for both binding experiments, which are shown beyond lane 5. (C) Interaction between bacterial RecA and RAD54B26–225. The binding experiment was performed in the presence of 100 mM KCl. The bands were visualized by Coomassie Brilliant Blue staining.
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Figure 3: RAD54B26–225 interacts with RAD51 and DMC1. The interactions were observed by a pull-down assay, in which DMC1 (A) or RAD51 (B) was mixed with RAD54B26–225 that was covalently conjugated to an Affi-Gel 15 matrix. The proteins bound to the RAD54B26–225-conjugated beads were eluted by SDS–PAGE sample buffer, and fractionated on a 12% SDS–PAGE gel. Lanes 2 and 3 are one-tenth of the total proteins used. Lane 4 is the negative control using the Affi-Gel 15 matrix without RAD54B26–225. The salt concentration was titrated for both binding experiments, which are shown beyond lane 5. (C) Interaction between bacterial RecA and RAD54B26–225. The binding experiment was performed in the presence of 100 mM KCl. The bands were visualized by Coomassie Brilliant Blue staining.

Mentions: We have previously shown that RAD54B interacts with RAD51 and DMC1 (30). However, it is not known whether the N-terminal domain of RAD54B is involved in the interactions. We therefore tested the interactions between the N-terminal domain of RAD54B and RAD51 or DMC1 by a pull-down assay, using RAD54B26–225-conjugated Affi-Gel 15 beads. The proteins bound to the RAD54B26–225 beads were detected by SDS–PAGE. Consistent with the fact that RAD54B26–225 self-associates (Figure 1C), we observed RAD54B26–225 in the elution fraction that was not covalently conjugated to the Affi-Gel beads (Figure 3A, lanes 5–9; 3B, lanes 5–8 and 3C, lane 5). As shown in Figures 3A and B, RAD54B26–225 interacted with both RAD51 and DMC1 (Figure 3A, lanes 5–9 and 3B, lanes 5 and 6, respectively). In contrast, RAD54B26–225 weakly bound to RecA, the bacterial homolog of RAD51 and DMC1, suggesting that the interactions between RAD54B26–225 and RAD51 or DMC1 were specific (Figure 3C). When the salt concentrations were titrated for the RAD51- and DMC1-binding experiments, the amounts of RAD51 and DMC1 bound to RAD54B26–225 sharply decreased at 200 mM of KCl (Figure 3A, lane 7 and 3B, lane 6). In the case of DMC1, the binding was observed even at 300 mM of KCl, whereas the RAD51 binding was absent at 250 mM of KCl. This difference in binding affinities could reflect the differences in the mechanisms of interactions between RAD54B26–225 and RAD51 or DMC1.Figure 3.


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)

RAD54B26–225 interacts with RAD51 and DMC1. The interactions were observed by a pull-down assay, in which DMC1 (A) or RAD51 (B) was mixed with RAD54B26–225 that was covalently conjugated to an Affi-Gel 15 matrix. The proteins bound to the RAD54B26–225-conjugated beads were eluted by SDS–PAGE sample buffer, and fractionated on a 12% SDS–PAGE gel. Lanes 2 and 3 are one-tenth of the total proteins used. Lane 4 is the negative control using the Affi-Gel 15 matrix without RAD54B26–225. The salt concentration was titrated for both binding experiments, which are shown beyond lane 5. (C) Interaction between bacterial RecA and RAD54B26–225. The binding experiment was performed in the presence of 100 mM KCl. The bands were visualized by Coomassie Brilliant Blue staining.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: RAD54B26–225 interacts with RAD51 and DMC1. The interactions were observed by a pull-down assay, in which DMC1 (A) or RAD51 (B) was mixed with RAD54B26–225 that was covalently conjugated to an Affi-Gel 15 matrix. The proteins bound to the RAD54B26–225-conjugated beads were eluted by SDS–PAGE sample buffer, and fractionated on a 12% SDS–PAGE gel. Lanes 2 and 3 are one-tenth of the total proteins used. Lane 4 is the negative control using the Affi-Gel 15 matrix without RAD54B26–225. The salt concentration was titrated for both binding experiments, which are shown beyond lane 5. (C) Interaction between bacterial RecA and RAD54B26–225. The binding experiment was performed in the presence of 100 mM KCl. The bands were visualized by Coomassie Brilliant Blue staining.
Mentions: We have previously shown that RAD54B interacts with RAD51 and DMC1 (30). However, it is not known whether the N-terminal domain of RAD54B is involved in the interactions. We therefore tested the interactions between the N-terminal domain of RAD54B and RAD51 or DMC1 by a pull-down assay, using RAD54B26–225-conjugated Affi-Gel 15 beads. The proteins bound to the RAD54B26–225 beads were detected by SDS–PAGE. Consistent with the fact that RAD54B26–225 self-associates (Figure 1C), we observed RAD54B26–225 in the elution fraction that was not covalently conjugated to the Affi-Gel beads (Figure 3A, lanes 5–9; 3B, lanes 5–8 and 3C, lane 5). As shown in Figures 3A and B, RAD54B26–225 interacted with both RAD51 and DMC1 (Figure 3A, lanes 5–9 and 3B, lanes 5 and 6, respectively). In contrast, RAD54B26–225 weakly bound to RecA, the bacterial homolog of RAD51 and DMC1, suggesting that the interactions between RAD54B26–225 and RAD51 or DMC1 were specific (Figure 3C). When the salt concentrations were titrated for the RAD51- and DMC1-binding experiments, the amounts of RAD51 and DMC1 bound to RAD54B26–225 sharply decreased at 200 mM of KCl (Figure 3A, lane 7 and 3B, lane 6). In the case of DMC1, the binding was observed even at 300 mM of KCl, whereas the RAD51 binding was absent at 250 mM of KCl. This difference in binding affinities could reflect the differences in the mechanisms of interactions between RAD54B26–225 and RAD51 or DMC1.Figure 3.

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