We have shown that the recently identified human Rad54 homolog, Rad54B, is a DNA-binding protein and a dsDNA-dependent ATPase such as hRad54. hRad54B shares homology with ScTid1/Rdh54, which is another Rad54 homolog in yeast, suggesting that hRad54B is the human counterpart of ScTid1/Rdh54. But unlike ScTid1/Rdh54, which interacts with yeast Rad51 and Dmc1, we could not detect the direct interaction of hRad54B with hRad51 and hDmc1, indicating the functional difference between ScTid1/Rdh54 and hRad54B.
was cloned by the sequence similarity with hRAD54
). ScRad54, ScTid1/Rdh54, hRad54 and hRad54B belong to a subfamily of the SNF2/SWI2 family because they share high homology not only in the seven helicase motifs but also around these motifs in the SNF2 domain (5
). There is a highly homologous region in the N-terminal domain of hRad54B and ScTid1/Rdh54, where little homology exists between hRad54 and hRad54B. This region contains a cluster of basic amino acid residues, which is a putative nuclear localization signal. We found that there are SNF2/SWI2 family proteins containing the similar region in other eukaryotes, indicating that the structure of ScTid1/Rdh54 is conserved among eukaryotes. These findings suggest that hRad54B is the human counterpart of ScTid1/Rdh54.
Human Rad54B was found to be a DNA-binding protein and to possess a dsDNA-dependent ATPase activity such as ScTid1/Rdh54, but there were two functional differences between hRad54B and ScTid1/Rdh54. First, hRad54B showed lower ATPase activity than did hRad54. In contrast to our results, previous studies have shown that the rate of ATP hydrolysis by ScRad54 and ScTid1/Rdh54 are comparable (1.27 × 103
at 1 mM ATP and 2.2 × 103
at 1.5 mM ATP, respectively) (7
). Since we compared the ATPase activities of hRad54 and hRad54B under the same conditions using the proteins purified by the same procedure, it is likely that hRad54B is a less active ATPase than hRad54, showing a difference between species. Second, neither hRad51 nor hDmc1 interacted directly with hRad54B. We have previously reported that hRad54B associates with hRad51 in mammalian cells (15
). One possibility is that hRad54B interacts with hRad51 indirectly and that there may be other proteins mediating the complex formation. Co-localization of hRad54B with hRad51, hRad54 and Brca1 in the nucleus supports the hypothesis that hRad54B is included in the recombination protein complex (15
). Another possibility is that some post-translational modification, such as phosphorylation, stimulates the interaction between hRad54B and hRad51 (or hDmc1), as previously reported concerning the interaction between Rad51 and Rad52 (28
, both Rad54 and Tid1/Rdh54 play roles in homologous recombination. It has been reported that ScTid1/Rdh54 functions preferentially in recombination between homologous chromosomes, while ScRad54 is required primarily for sister chromatid-based repair (17
). Human Rad54 has similar biochemical properties to its yeast counterpart and acts as a recombination protein. Biochemically, hRad54 possesses a robust ATPase activity and interacts with hRad51 (23
), which enhances the ATPase activity of hRad54. Biologically, homozygous RAD54
mutants in mouse and chicken show high sensitivity to radiation and methyl methanesulfonate and have reduced levels of homologous recombination (13
). In contrast, it is not clear whether hRad54B is involved in homologous recombination. But we have found that inactivation of hRAD54B
in a colon cancer cell line resulted in severe reduction of targeted integration frequency (29
), suggesting that hRAD54B
also plays a role in homologous recombination. It is of interest whether hRad54B plays a role in meiotic recombination and mitotic interchromosomal recombination, which is considered to be very rare in animal cells.
Recently, the yeast SWI/SNF complex and other SWI2/SNF2 family protein-containing complexes were shown to generate superhelical torsion in DNA and chromatin (30
), which may disrupt chromatin structure and increase the accessibility of DNA within chromatin. It was reported that human and yeast Rad54 alter DNA superhelicity using the energy of ATP hydrolysis (8
). The dsDNA-dependent ATPase activities of hRad54 and hRad54B may play a role in the recombination process by making chromatin structure accessible for other recombination proteins or nucleoprotein filaments.
epistasis group genes are well conserved throughout evolution, but recent studies revealed that the role of each gene product is not necessarily the same in vertebrates as compared with yeast (2
). For example, Rad52 is essential in yeast, while Rad52-deficient vertebrate cells show subtle phenotype (32
), although DT40 cells deficient in both Rad52 and Xrcc3 are not viable (34
). It has also been reported that the Rad51-paralog proteins, Xrcc3 and Rad51C, form a stable complex and catalyze the D-loop formation (35
). Both ScRad54 and ScTid1/Rdh54 enhance the D-loop formation by ScRad51 (7
). We have examined the effects of hRad54 and hRad54B on the D-loop formation by hRad51 and hDmc1 using the pGsat4 plasmid and the 50mer oligonucleotide, SAT-1, as substrates (22
), but neither hRad54 nor hRad54B enhanced the D-loop formation by hRad51 and hDmc1 (K.Tanaka, W.Kagawa, T.Kinebuchi, H.Kurumizaka and K.Miyagawa, unpublished data). These findings suggest that hRad54 and hRad54B behave in a different way from ScRad54 and ScTid1/Rdh54 in the recombination process.
We have reported mutations in hRAD54
in primary tumors (5
). It is important to study whether these mutations affect the biochemical and biological functions of hRad54 and hRad54B. Recently, Lee et al
. reported that ScTid1/Rdh54 is required for adaptation from G2
/M arrest induced by a double-strand break in yeast (37
), suggesting that hRad54B might play a role separate from homologous recombination in mitotic cells. Further investigations are required for a comprehensive understanding of the functions of hRad54 and hRad54B, and their roles in carcinogenesis.