Phage Orf family recombinases: conservation of activities and involvement of the central channel in DNA binding

Curtis, F.A.; Malay, A.D.; Trotter, A.J.; Wilson, L.A.; Barradell-Black, M.M.; Bowers, L.Y.; Reed, P.; Hillyar, C.R.T.; Yeo, R.P.; Sanderson, J.M.; Heddle, J.G.; Sharples, G.J.

Abstract

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.

Citation

Curtis, F., Malay, A., Trotter, A., Wilson, L., Barradell-Black, M., Bowers, L., …Sharples, G. (2014). Phage Orf family recombinases: conservation of activities and involvement of the central channel in DNA binding. PLoS ONE, 9(8), Article e102454. https://doi.org/10.1371/journal.pone.0102454

• Journal Article Type: Article
• Acceptance Date: Jun 17, 2014
• Online Publication Date: Aug 1, 2014
• Publication Date: Aug 1, 2014
• Deposit Date: May 9, 2014
• Publicly Available Date: Aug 6, 2014
• Journal: PLoS ONE
• Publisher: Public Library of Science
• Peer Reviewed: Peer Reviewed
• Volume: 9
• Issue: 8
• Article Number: e102454
• DOI: https://doi.org/10.1371/journal.pone.0102454
• Public URL: https://durham-repository.worktribe.com/output/1455398