The C-terminus of the phage λ Orf recombinase is involved in DNA binding

Curtis, F. A.; Reed, P.; Wilson, L. A.; Bowers, L. Y.; Yeo, R. P.; Sanderson, J. M.; Walmsley, A. R.; Sharples, G. J.

Abstract

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf–SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.

Citation

Curtis, F. A., Reed, P., Wilson, L. A., Bowers, L. Y., Yeo, R. P., Sanderson, J. M., Walmsley, A. R., & Sharples, G. J. (2011). The C-terminus of the phage λ Orf recombinase is involved in DNA binding. Journal of Molecular Recognition, 24(2), 333-340. https://doi.org/10.1002/jmr.1079

• Journal Article Type: Article
• Online Publication Date: Dec 13, 2010
• Publication Date: Mar 1, 2011
• Deposit Date: Jan 25, 2013
• Journal: Journal of Molecular Recognition
• Print ISSN: 0952-3499
• Electronic ISSN: 1099-1352
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 24
• Issue: 2
• Pages: 333-340
• DOI: https://doi.org/10.1002/jmr.1079
• Keywords: Genetic recombination, DNA repair, Bacteriophage λ, NinB, RecFOR, SSB.
• Public URL: https://durham-repository.worktribe.com/output/1490282