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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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Proposed orientations of the RAD54B26–225-interacting regions of DMC1 in the helical filament form. DMC1 sites essential for the interaction with RAD54B26–225 were mapped on the corresponding locations of the M. voltae RadA filament. The closeup view shows the ATP-binding site that is surrounded by the RAD54B26–225-interacting regions. All structural figures were prepared using the PyMOL program (44).
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Figure 6: Proposed orientations of the RAD54B26–225-interacting regions of DMC1 in the helical filament form. DMC1 sites essential for the interaction with RAD54B26–225 were mapped on the corresponding locations of the M. voltae RadA filament. The closeup view shows the ATP-binding site that is surrounded by the RAD54B26–225-interacting regions. All structural figures were prepared using the PyMOL program (44).

Mentions: The RAD54B26–225 fragment also physically interacted with both the RAD51 and DMC1 recombinases. Previously, we demonstrated that RAD54B stimulates the DMC1-mediated strand exchange by stabilizing the DMC1–ssDNA complex, and proposed that RAD54B may promote the formation of the active DMC1 helical filament (30). In this study, we found that RAD54B26–225 bound to both DMC1 alone and DMC1 complexed with DNA, and we mapped the RAD54B-interacting regions of DMC1 (amino acid residues 153–214 and 296–340). These regions are exposed on the surface of the octameric ring. The corresponding regions in the Methanococcus voltae RadA protein, a homolog of DMC1 that forms a helical filament (43), are also exposed on the surface. Thus, the RAD54B-interacting regions appear to be easily accessible by other factors, in both the ring and helical filament forms. These regions, for example, do not overlap with the putative DNA-binding loops L1 and L2 (amino acid residues 235–240 and 271–286) that face towards the center of the ring or the filament structure. This fact is consistent with the results that RAD54B26–225 interacted with the DMC1 bound to either ssDNA or dsDNA. Interestingly, the corresponding RAD54B-interacting regions of RadA are located near the monomer–monomer interface, and contain amino acid residues that directly interact with ATP (Figure 6). Thus, the N-terminal region of RAD54B may affect the quaternary structure of DMC1 through these interactions, and may be critical for regulating the function of DMC1. Given that RAD54B stimulates the DMC1-mediated DNA strand exchange, one possibility is that the binding of RAD54B to DMC1 may trigger the conversion of the DMC1 structure from the octameric ring form to the active helical filament form. Another possibility is that the binding of RAD54B to DMC1 may promote the turnover of DMC1 from the DNA strand-exchange product, leading to the release of DMC1. Although these observations suggested that the interaction between RAD54B and DMC1 is functionally important, meiosis in the RAD54B knockout mouse seems to be unaffected (29). This may be due to the presence of an unidentified RAD54 paralog that functions in meiosis. Alternatively, RAD54B may have a relatively minor role in meiosis, and may function with DMC1 only under certain circumstances. Further in vivo and in vitro analyses are required to elucidate the role of RAD54B in meiosis.Figure 6.


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)

Proposed orientations of the RAD54B26–225-interacting regions of DMC1 in the helical filament form. DMC1 sites essential for the interaction with RAD54B26–225 were mapped on the corresponding locations of the M. voltae RadA filament. The closeup view shows the ATP-binding site that is surrounded by the RAD54B26–225-interacting regions. All structural figures were prepared using the PyMOL program (44).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Proposed orientations of the RAD54B26–225-interacting regions of DMC1 in the helical filament form. DMC1 sites essential for the interaction with RAD54B26–225 were mapped on the corresponding locations of the M. voltae RadA filament. The closeup view shows the ATP-binding site that is surrounded by the RAD54B26–225-interacting regions. All structural figures were prepared using the PyMOL program (44).
Mentions: The RAD54B26–225 fragment also physically interacted with both the RAD51 and DMC1 recombinases. Previously, we demonstrated that RAD54B stimulates the DMC1-mediated strand exchange by stabilizing the DMC1–ssDNA complex, and proposed that RAD54B may promote the formation of the active DMC1 helical filament (30). In this study, we found that RAD54B26–225 bound to both DMC1 alone and DMC1 complexed with DNA, and we mapped the RAD54B-interacting regions of DMC1 (amino acid residues 153–214 and 296–340). These regions are exposed on the surface of the octameric ring. The corresponding regions in the Methanococcus voltae RadA protein, a homolog of DMC1 that forms a helical filament (43), are also exposed on the surface. Thus, the RAD54B-interacting regions appear to be easily accessible by other factors, in both the ring and helical filament forms. These regions, for example, do not overlap with the putative DNA-binding loops L1 and L2 (amino acid residues 235–240 and 271–286) that face towards the center of the ring or the filament structure. This fact is consistent with the results that RAD54B26–225 interacted with the DMC1 bound to either ssDNA or dsDNA. Interestingly, the corresponding RAD54B-interacting regions of RadA are located near the monomer–monomer interface, and contain amino acid residues that directly interact with ATP (Figure 6). Thus, the N-terminal region of RAD54B may affect the quaternary structure of DMC1 through these interactions, and may be critical for regulating the function of DMC1. Given that RAD54B stimulates the DMC1-mediated DNA strand exchange, one possibility is that the binding of RAD54B to DMC1 may trigger the conversion of the DMC1 structure from the octameric ring form to the active helical filament form. Another possibility is that the binding of RAD54B to DMC1 may promote the turnover of DMC1 from the DNA strand-exchange product, leading to the release of DMC1. Although these observations suggested that the interaction between RAD54B and DMC1 is functionally important, meiosis in the RAD54B knockout mouse seems to be unaffected (29). This may be due to the presence of an unidentified RAD54 paralog that functions in meiosis. Alternatively, RAD54B may have a relatively minor role in meiosis, and may function with DMC1 only under certain circumstances. Further in vivo and in vitro analyses are required to elucidate the role of RAD54B in meiosis.Figure 6.

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