tailieunhanh - Báo cáo khoa học: Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance

The crystal structure of theSchizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant struc-tural homology with the Escherichia coliresolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in of thea-helices that form this bundle show homology to a putative DNA-binding motif known as investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in fission yeast. | Eur. J. Biochem. 270 2837-2847 2003 FEBS 2003 doi Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 in vivo role in mitochondrial DNA maintenance Barbara Sigala and Irina R. Tsaneva Department of Biochemistry and Molecular Biology University College London London UK The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the a-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To mvettigate the biochemical lunchon of the triple-helix domain truncated Ydc2 mutants were expressed in E. coll and in fission yeast. Although the tnm-cated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to syntheticjunctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif Ceschini et al. 2001 EMBO J. 20 6601-6611 . The-biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Dydc2 S. pmmee. The tuiins-ated muuSonss were hhown oo localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. PSasmiids CẦUI- ull-lh i jufft Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA phenotype . These reurtss provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pmmbe. Keywords Holliday junction resolvase

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