Crp1p, a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae.
A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.
Rass
Ulrich
U
Institut für Genetik der Universität zu Köln, Zülpicher Strasse 47, Köln, Germany. uli.rass@uni-koeln.de
Kemper
Börries
B
D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D008565:Membrane Proteins; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
C468650:CRP1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C102153:MDG1 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII