Importance of minor-groove contacts for the recognition of DNA by the binding domain of Hin recombinase

Abstract

Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.

The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development.

“…4D and 6A) showed that methylation of residues in either the major or minor groove inhibited AbaA binding, supporting the conclusion that the protein makes essential contacts in both grooves. Only a few other proteins make essential contacts in both grooves (4,40,46). Furthermore, this observation is consistent with the proposed interactions between 3-sheets and the minor groove of DNA (16 (52).…”

Structure-guided reprogramming of serine recombinase DNA sequence specificity

“…1A). The interactions between the minor groove of substrate DNA and the γδ resolvase arm region residues Arg 125, Thr 126, Gly 129, Arg 130, Phe 140, Gly 141, and Arg 142 provide the structural and functional foundation for DNA association (31,32). Comparative sequence analysis of functionally divergent resolvases and invertases and their cognate DNA binding sites suggests that variable arm region positions may also provide the basis for catalytic domain sequence specificity (Fig.…”

Role of arginine‐43 and arginine‐69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

“…Previous studies demonstrated that a synthetic peptide ᮊ 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 1235-1247 consisting of residues 139-190 (52-amino-acid peptide) of Hin bound specifically to a hix half-site (Sluka et al, 1990). The difference between the binding affinity of the 52-amino-acid peptide (K d Ϸ10 ¹7 ) and the wild-type Hin dimer (K d Ϸ10 ¹9 ) was partly attributed to the co-operative interactions between the amino-terminal domains required for dimer formation (Bruist et al, 1987b;Sluka et al, 1987;Glasgow et al, 1989a).…”

“…A C-terminal 52-amino-acid polypeptide of Hin recombinase (residues 139-190) has been shown to contain the DNA-binding domain (Sluka et al, 1987). Biochemical and crystallographic data have demonstrated that a helix-turn-helix structure (residues 149 to 180) makes specific contacts in the major groove with nucleotides at positions 9 and 10 of the hix half-site (Glasgow et al, 1989a;Sluka et al, 1990;Hughes et al, 1992;Feng et al, 1994b) (Fig. 2).…”