Crp1p, a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

Rass, Ulrich and Kemper, Börries (2002) Crp1p, a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae. Journal of Molecular Biology, 323 (4). pp. 685-700. ISSN 0022-2836

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Abstract

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.

Item Type: Article
Keywords: Holliday junction, Holliday junction resolution, DNA-binding protein, homologous recombination, cruciform DNA, DNA repair
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Research Centres and Groups: Genome Damage and Stability Centre
Subjects: Q Science > Q Science (General) > Q0179.9 Research
Q Science > Q Science (General)
Depositing User: Ulrich Rass
Date Deposited: 26 Sep 2018 09:28
Last Modified: 26 Sep 2018 09:28
URI: http://srodev.sussex.ac.uk/id/eprint/79019
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