Rights statement: © 2014 Curtis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Phage Orf family recombinases
T2 - conservation of activities and involvement of the central channel in DNA binding
AU - Curtis, Fiona
AU - Malay, Ali D.
AU - Trotter, Alexander J.
AU - Wilson, Lindsay A.
AU - Barradell-Black, Michael M.
AU - Bowers, Laura Y.
AU - Reed, Patricia
AU - Hillyar, Christopher R. T.
AU - Yeo, Robert P.
AU - Sanderson, John M.
AU - Heddle, Jonathan G.
AU - Sharples, Gary J.
N1 - © 2014 Curtis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2014/8/1
Y1 - 2014/8/1
N2 - 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.
AB - 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.
U2 - 10.1371/journal.pone.0102454
DO - 10.1371/journal.pone.0102454
M3 - Journal article
VL - 9
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 8
M1 - e102454
ER -