Role of ATP-binding motifs on DNA-binding activity and biological function of Rhp51, a Rad51 homologue in fission yeast.
Rhp51, a RecA and Rad51 homologue of Schizosaccharomyces pombe, plays a pivotal role in homologous recombination and recombinational repair. It has a set of the well-conserved type A and type B ATP-binding motifs, which are highly conserved in all RecA homologues. In a previous study [Kim, Lee, Park, Park and Park (2001) Nucleic Acids Res. 29, 1724-1732], we reported that a single mutation of the conserved lysine in A motif [Lys(155)-->Ala (K155A)] destroyed the DNA repair ability of Rhp51 and that overexpression of this mutant protein conferred dominant negativity. In the present paper, we investigated DNA-binding properties of recombinant Rhp51 and its mutant proteins. Purified Rhp51 protein showed ATP-dependent double- and single-strand DNA-binding activities. To characterize the role of ATP-binding motifs, we generated Rhp51 K155A and Rhp51 Asp(244)-->Gln (D244Q), which have a single amino acid substitution in A and B motifs respectively. Interestingly, K155A and D244Q mutations impaired ATP-dependent DNA binding in a different manner. K155A lost the DNA binding itself, whereas D244Q maintained the binding ability but lost the ATP dependency. However, despite the difference in DNA-binding ability, both mutations failed to rescue the methylmethane sulphonate and UV sensitivity of the rhp51Delta mutant. Together, these results suggested that not only the DNA binding but also the ATP dependence in DNA binding is required for proper in vivo functioning of Rhp51.
Kim
Woo J
WJ
School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
Lee
Hyojin
H
Park
Eon J
EJ
Hong
Seung H
SH
Park
Sang D
SD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D017124:Conserved Sequence; D017931:DNA Primers; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000378:metabolism; D051135:Rad51 Recombinase; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C084232:RHP51 protein, S pombe; D016601:RNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; D000255:Adenosine Triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D008239:Lysine