title: Phage ORF family recombinases: conservation of activities and involvement of the central channel in DNA binding.
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.
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Curtis
authors.affiliation: Heddle Initiative Research Unit, RIKEN, Wako, Saitama, Japan.
authors.forename: Ali D
authors.initials: AD
authors.lastname: Malay
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Trotter
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Lindsay A
authors.initials: LA
authors.lastname: Wilson
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Michael M H
authors.initials: MM
authors.lastname: Barradell-Black
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Laura Y
authors.initials: LY
authors.lastname: Bowers
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Patricia
authors.initials: P
authors.lastname: Reed
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Christopher R T
authors.initials: CR
authors.lastname: Hillyar
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Yeo
authors.affiliation: Department of Chemistry, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: John M
authors.initials: JM
authors.lastname: Sanderson
authors.affiliation: Heddle Initiative Research Unit, RIKEN, Wako, Saitama, Japan.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Heddle
authors.affiliation: School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Sharples
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins; D023061:Gene Order; D016679:Genome, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D045522:Recombinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D045522:Recombinases; D014764:Viral Proteins
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