DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein.
The AT motif-binding factor 1 (ATBF1)-A is a large transcription factor containing four homeodomains and 23 zinc finger motifs. It has a number of motifs involved in transcriptional regulation, and in addition, several motifs found in enzymes, such as ATPases and helicases. In this study, we examined whether ATPase activity is associated with the ATBF1-A molecule. A 263-amino acid segment of the ATBF1-A molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in Escherichia coli in the form of glutathione S-transferase fusion protein and analyzed for ATPase activity. We found that AHZ was able to hydrolyze ATP with K(m) 10.6 microM and K(cat) 0.055 min(-1) at 5 mM Mg(2+) and pH 7.75. AHZ retained bacterial DNA and removal of the DNA resulted in 70% decrease in ATPase activity. The addition of double- or single-stranded DNAs restored 70-75% ATPase activity and that of RNA restored 50-55% activity. Site-directed mutagenesis of the A-motif resulted in 34% reduction of ATPase activity with no significant loss of bound DNA. In contrast, mutation of homeodomain IV and zinc finger 21 resulted in 90 and 80% reduction of ATPase, respectively, with the loss of the ability to bind to DNA and RNA. These results show that ATBF1 has at least one enzyme activity in addition to regulation of DNA transcription. The ATPase activity associated with ATBF1-A is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs.
Kawaguchi
M
M
Department of Pathology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. kawaguch@ms.toyama-mpu.ac.jp
Miura
Y
Y
Ido
A
A
Morinaga
T
T
Sakata
N
N
Oya
T
T
Hashimoto-Tamaoki
T
T
Sasahara
M
M
Koizumi
F
F
Tamaoki
T
T
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D012313:RNA / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D016335:Zinc Fingers
D004269:DNA, Bacterial; D018398:Homeodomain Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C037693:single stranded DNA dependent ATPase