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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) Sequence comparison of RAD54 and RAD54B. These proteins are separated into three regions (N-terminal domain, SWI2/SNF2 domain and C-terminal domain), and the amino acid sequence identities and similarities between these proteins were calculated for each region. The amino acid number at the boundary of each domain is denoted. The gray lines indicate the seven helicase motifs (I, Ia, II, III, IV, V and VI, respectively). (B) Purification of the RAD54B26–225 protein. The peak fractions from the Ni-NTA agarose column (lane 2), the fraction after the removal of the His6 tag (lane 3), the Benzamidine Sepharose flow-through (lane 4), the Q-Sepharose flow-through (lane 5) and the peak fractions from the SP-Sepharose column (lane 6) were analyzed on a 12% SDS–PAGE gel, which was stained with Coomassie Brilliant Blue. Lane 1 indicates the molecular mass markers. (C) Gel filtration analysis of RAD54B26–225. The arrow indicates the peak location of a molecular weight marker, ovalbumin (43 kDa), which nearly corresponds to that of RAD54B26–225.
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Figure 1: (A) Sequence comparison of RAD54 and RAD54B. These proteins are separated into three regions (N-terminal domain, SWI2/SNF2 domain and C-terminal domain), and the amino acid sequence identities and similarities between these proteins were calculated for each region. The amino acid number at the boundary of each domain is denoted. The gray lines indicate the seven helicase motifs (I, Ia, II, III, IV, V and VI, respectively). (B) Purification of the RAD54B26–225 protein. The peak fractions from the Ni-NTA agarose column (lane 2), the fraction after the removal of the His6 tag (lane 3), the Benzamidine Sepharose flow-through (lane 4), the Q-Sepharose flow-through (lane 5) and the peak fractions from the SP-Sepharose column (lane 6) were analyzed on a 12% SDS–PAGE gel, which was stained with Coomassie Brilliant Blue. Lane 1 indicates the molecular mass markers. (C) Gel filtration analysis of RAD54B26–225. The arrow indicates the peak location of a molecular weight marker, ovalbumin (43 kDa), which nearly corresponds to that of RAD54B26–225.

Mentions: To gain insight into the function of the N-terminal region of RAD54B, which is less conserved between RAD54 and RAD54B (Figure 1A), we constructed a RAD54B deletion mutant containing the first 295 residues (RAD54B1–295). However, this fragment rapidly degraded to several smaller fragments during the expression and purification processes, suggesting that the fragment contained unstructured or flexible regions. Several rounds of fragment design and purification were performed to identify the stable N-terminal region of RAD54B. We found that the fragment consisting of amino acid residues 26–225 of RAD54B (RAD54B26–225) was resistant to proteolysis and was highly soluble. The RAD54B26–225 mutant was expressed in the E. coli JM109 (DE3) strain, as a fusion protein with an N-terminal His6 tag containing a cleavage site for thrombin protease, and was purified by Ni-NTA column chromatography (Figure 1B, lane 2). After the His6 tag was uncoupled with thrombin protease (Figure 1B, lane 3), the peak fractions containing RAD54B26–225 were further purified by Benzamidine column chromatography (Figure 1B, lane 4), Q-Sepharose column chromatography (Figure 1B, lane 5) and SP-Sepharose column chromatography (Figure 1B, lane 6). About 10 mg of purified RAD54B26–225 were obtained from 2.5 l of E. coli suspension culture. The SDS–PAGE analysis of the final purification fraction revealed an additional band with an apparent molecular weight of about 50 kDa. The band disappeared when higher concentrations of reducing agent were included in the electrophoresis sample buffer, indicating that RAD54B26–225 oligomerizes. Consistent with this observation, a gel filtration analysis of the purified RAD54B26–225 indicated that the fragment primarily exists as a dimer (Figure 1C). These results demonstrated that amino acid residues 26–225 of RAD54B form a stable domain. Although it is not known whether the full-length RAD54B protein multimerizes, the N-terminal region may play a role in the self-association of RAD54B.Figure 1.


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) Sequence comparison of RAD54 and RAD54B. These proteins are separated into three regions (N-terminal domain, SWI2/SNF2 domain and C-terminal domain), and the amino acid sequence identities and similarities between these proteins were calculated for each region. The amino acid number at the boundary of each domain is denoted. The gray lines indicate the seven helicase motifs (I, Ia, II, III, IV, V and VI, respectively). (B) Purification of the RAD54B26–225 protein. The peak fractions from the Ni-NTA agarose column (lane 2), the fraction after the removal of the His6 tag (lane 3), the Benzamidine Sepharose flow-through (lane 4), the Q-Sepharose flow-through (lane 5) and the peak fractions from the SP-Sepharose column (lane 6) were analyzed on a 12% SDS–PAGE gel, which was stained with Coomassie Brilliant Blue. Lane 1 indicates the molecular mass markers. (C) Gel filtration analysis of RAD54B26–225. The arrow indicates the peak location of a molecular weight marker, ovalbumin (43 kDa), which nearly corresponds to that of RAD54B26–225.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: (A) Sequence comparison of RAD54 and RAD54B. These proteins are separated into three regions (N-terminal domain, SWI2/SNF2 domain and C-terminal domain), and the amino acid sequence identities and similarities between these proteins were calculated for each region. The amino acid number at the boundary of each domain is denoted. The gray lines indicate the seven helicase motifs (I, Ia, II, III, IV, V and VI, respectively). (B) Purification of the RAD54B26–225 protein. The peak fractions from the Ni-NTA agarose column (lane 2), the fraction after the removal of the His6 tag (lane 3), the Benzamidine Sepharose flow-through (lane 4), the Q-Sepharose flow-through (lane 5) and the peak fractions from the SP-Sepharose column (lane 6) were analyzed on a 12% SDS–PAGE gel, which was stained with Coomassie Brilliant Blue. Lane 1 indicates the molecular mass markers. (C) Gel filtration analysis of RAD54B26–225. The arrow indicates the peak location of a molecular weight marker, ovalbumin (43 kDa), which nearly corresponds to that of RAD54B26–225.
Mentions: To gain insight into the function of the N-terminal region of RAD54B, which is less conserved between RAD54 and RAD54B (Figure 1A), we constructed a RAD54B deletion mutant containing the first 295 residues (RAD54B1–295). However, this fragment rapidly degraded to several smaller fragments during the expression and purification processes, suggesting that the fragment contained unstructured or flexible regions. Several rounds of fragment design and purification were performed to identify the stable N-terminal region of RAD54B. We found that the fragment consisting of amino acid residues 26–225 of RAD54B (RAD54B26–225) was resistant to proteolysis and was highly soluble. The RAD54B26–225 mutant was expressed in the E. coli JM109 (DE3) strain, as a fusion protein with an N-terminal His6 tag containing a cleavage site for thrombin protease, and was purified by Ni-NTA column chromatography (Figure 1B, lane 2). After the His6 tag was uncoupled with thrombin protease (Figure 1B, lane 3), the peak fractions containing RAD54B26–225 were further purified by Benzamidine column chromatography (Figure 1B, lane 4), Q-Sepharose column chromatography (Figure 1B, lane 5) and SP-Sepharose column chromatography (Figure 1B, lane 6). About 10 mg of purified RAD54B26–225 were obtained from 2.5 l of E. coli suspension culture. The SDS–PAGE analysis of the final purification fraction revealed an additional band with an apparent molecular weight of about 50 kDa. The band disappeared when higher concentrations of reducing agent were included in the electrophoresis sample buffer, indicating that RAD54B26–225 oligomerizes. Consistent with this observation, a gel filtration analysis of the purified RAD54B26–225 indicated that the fragment primarily exists as a dimer (Figure 1C). These results demonstrated that amino acid residues 26–225 of RAD54B form a stable domain. Although it is not known whether the full-length RAD54B protein multimerizes, the N-terminal region may play a role in the self-association of RAD54B.Figure 1.

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