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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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(A) A schematic representation of the 10 overlapping GST–DMC1 fusion proteins. The gray bar indicates the N-terminal domain of DMC1, and the white bar indicates the core ATPase domain. (B) Protein–protein interaction assay of RAD54B26–225 with the DMC1 deletion mutants. The GS4B–DMC1 deletion mutant beads were first mixed with BSA, to prevent nonspecific protein binding, followed by the addition of RAD54B26–225. After an incubation at 4°C for 1 h, the GS4B–DMC1 deletion mutant beads were washed with binding buffer. The RAD54B26–225 proteins that bound to the GS4B–DMC1 deletion mutant beads were fractionated by 12% SDS–PAGE gel (lanes 2–11, respectively). Lane 1 is one-tenth of the input proteins, and lane 12 is the negative control experiment using the GS4B beads without the DMC1 deletion mutant. (C) The RAD54B26–225-binding sites mapped on the DMC1 octameric ring. The purple region indicates DMC1153–214, and the yellow region indicates DMC1296–340.
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Figure 5: (A) A schematic representation of the 10 overlapping GST–DMC1 fusion proteins. The gray bar indicates the N-terminal domain of DMC1, and the white bar indicates the core ATPase domain. (B) Protein–protein interaction assay of RAD54B26–225 with the DMC1 deletion mutants. The GS4B–DMC1 deletion mutant beads were first mixed with BSA, to prevent nonspecific protein binding, followed by the addition of RAD54B26–225. After an incubation at 4°C for 1 h, the GS4B–DMC1 deletion mutant beads were washed with binding buffer. The RAD54B26–225 proteins that bound to the GS4B–DMC1 deletion mutant beads were fractionated by 12% SDS–PAGE gel (lanes 2–11, respectively). Lane 1 is one-tenth of the input proteins, and lane 12 is the negative control experiment using the GS4B beads without the DMC1 deletion mutant. (C) The RAD54B26–225-binding sites mapped on the DMC1 octameric ring. The purple region indicates DMC1153–214, and the yellow region indicates DMC1296–340.

Mentions: Previously, we found that RAD54B interacts with the ATPase domain of DMC1. To define more precisely the regions of DMC1 that interact with RAD54B, 10 DMC1 fragments were designed to cover the entire region of the DMC1 sequence (Figure 5A). These fragments were expressed as GST-fused proteins. The GST-fused DMC1 fragments required a short induction time and rapid purification. Otherwise, the fragments readily degraded to a size of about 25 kDa, which is likely GST. Even with careful purification, partial degradation products were observed with some of the DMC1 fragments (Figure 5B, lanes 3, 4, 5, 7–9). A pull-down assay using GS4B beads was carried out (Figure 5B). In this assay, RAD54B26–225 was pulled down with GST-fused DMC1 fragments bound to GS4B beads, and was detected by SDS–PAGE. As shown in Figure 5B, RAD54B26–225 bound to DMC1 fragments VI and X, and weakly to V (Figure 5B, lanes 6, 7 and 11), but did not bind to other fragments. Regions VI and X are located close to each other and are exposed on the surface of the crystal structure of DMC1 (Figure 5C). The RAD54B26–225 bound to DMC1 fragments VI and X with relatively high affinity, and these DMC1 fragments were relatively stable, suggesting that the interactions are specific.Figure 5.


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)

(A) A schematic representation of the 10 overlapping GST–DMC1 fusion proteins. The gray bar indicates the N-terminal domain of DMC1, and the white bar indicates the core ATPase domain. (B) Protein–protein interaction assay of RAD54B26–225 with the DMC1 deletion mutants. The GS4B–DMC1 deletion mutant beads were first mixed with BSA, to prevent nonspecific protein binding, followed by the addition of RAD54B26–225. After an incubation at 4°C for 1 h, the GS4B–DMC1 deletion mutant beads were washed with binding buffer. The RAD54B26–225 proteins that bound to the GS4B–DMC1 deletion mutant beads were fractionated by 12% SDS–PAGE gel (lanes 2–11, respectively). Lane 1 is one-tenth of the input proteins, and lane 12 is the negative control experiment using the GS4B beads without the DMC1 deletion mutant. (C) The RAD54B26–225-binding sites mapped on the DMC1 octameric ring. The purple region indicates DMC1153–214, and the yellow region indicates DMC1296–340.
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Related In: Results  -  Collection

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

Figure 5: (A) A schematic representation of the 10 overlapping GST–DMC1 fusion proteins. The gray bar indicates the N-terminal domain of DMC1, and the white bar indicates the core ATPase domain. (B) Protein–protein interaction assay of RAD54B26–225 with the DMC1 deletion mutants. The GS4B–DMC1 deletion mutant beads were first mixed with BSA, to prevent nonspecific protein binding, followed by the addition of RAD54B26–225. After an incubation at 4°C for 1 h, the GS4B–DMC1 deletion mutant beads were washed with binding buffer. The RAD54B26–225 proteins that bound to the GS4B–DMC1 deletion mutant beads were fractionated by 12% SDS–PAGE gel (lanes 2–11, respectively). Lane 1 is one-tenth of the input proteins, and lane 12 is the negative control experiment using the GS4B beads without the DMC1 deletion mutant. (C) The RAD54B26–225-binding sites mapped on the DMC1 octameric ring. The purple region indicates DMC1153–214, and the yellow region indicates DMC1296–340.
Mentions: Previously, we found that RAD54B interacts with the ATPase domain of DMC1. To define more precisely the regions of DMC1 that interact with RAD54B, 10 DMC1 fragments were designed to cover the entire region of the DMC1 sequence (Figure 5A). These fragments were expressed as GST-fused proteins. The GST-fused DMC1 fragments required a short induction time and rapid purification. Otherwise, the fragments readily degraded to a size of about 25 kDa, which is likely GST. Even with careful purification, partial degradation products were observed with some of the DMC1 fragments (Figure 5B, lanes 3, 4, 5, 7–9). A pull-down assay using GS4B beads was carried out (Figure 5B). In this assay, RAD54B26–225 was pulled down with GST-fused DMC1 fragments bound to GS4B beads, and was detected by SDS–PAGE. As shown in Figure 5B, RAD54B26–225 bound to DMC1 fragments VI and X, and weakly to V (Figure 5B, lanes 6, 7 and 11), but did not bind to other fragments. Regions VI and X are located close to each other and are exposed on the surface of the crystal structure of DMC1 (Figure 5C). The RAD54B26–225 bound to DMC1 fragments VI and X with relatively high affinity, and these DMC1 fragments were relatively stable, suggesting that the interactions are specific.Figure 5.

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