Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent homologous pairing protein.
The Mhr1 protein is necessary for mtDNA homologous recombination in Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction during homologous recombination, and is generally catalyzed by the RecA/Rad51 family of proteins in an ATP-dependent manner. Mhr1 catalyzes HP through a mechanism similar, at the DNA level, to that of the RecA/Rad51 proteins, but without utilizing ATP. However, it has no sequence homology with the RecA/Rad51 family proteins or with other ATP-independent HP proteins, and exhibits different requirements for DNA topology. We are interested in the structural features of the functional domains of Mhr1. In this study, we employed the native fluorescence of Mhr1's Trp residues to examine the energy transfer from the Trp residues to etheno-modified ssDNA bound to Mhr1. Our results showed that two of the seven Trp residues (Trp71 and Trp165) are spatially close to the bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that Asp69 is involved in Mg(2+)-dependent DNA binding, and that multiple Lys and Arg residues located around Trp71 and Trp165 are involved in the DNA-binding activity of Mhr1. In addition, in vivo complementation analyses showed that a region around Trp165 is important for the maintenance of mtDNA. On the basis of these results, we discuss the function of the region surrounding Trp165.
Graduate School of Nanobioscience, Yokohama City University, Japan.
Tokiha
T
Masuda
Feng
F
Ling
Takehiko
T
Shibata
Tsutomu
T
Mikawa
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017385:Sequence Homology; D040681:Structural Homology, Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C417727:MHR1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D004247:DNA