ABSTRACT
We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15â bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriophage lambda/genetics , Bacteriophage lambda/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Bacterial Proteins/chemistry , Base Sequence , Binding Sites , Computer Simulation , DNA/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Factor For Inversion Stimulation Protein/chemistry , Factor For Inversion Stimulation Protein/genetics , Gene Expression , Mutant Proteins/metabolism , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Multimerization/genetics , Viral Proteins/chemistryABSTRACT
Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redß 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.
Subject(s)
Bacteriophages/genetics , Bacteriophages/metabolism , Multigene Family , Recombinases/genetics , Recombinases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Amino Acid Sequence , DNA, Single-Stranded/metabolism , DNA-Binding Proteins , Gene Order , Genome, Viral , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinases/chemistry , Sequence Alignment , Viral Proteins/chemistryABSTRACT
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.
