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.

Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction withEscherichia colisingle stranded DNA binding protein

“…Secondary structure elements above and below are for the CTD and Orf, respectively. The sequence of YbcN is also included in the alignment, based on a previous multiple sequence alignment of Orf homologs (51). Residues of Redβ that contact λ Exo are shown in bold red.…”

Systematic classification of the His-Me finger superfamily

“…Group 19 contains proteins from the phage Orf family of recombinases from Listeria phages (accession: CAC96489). Listeria phage members of the Orf family were indicated to be distantly related versions of a canonical Orf representatives and may constitute novel single-strand specific DNases that could have arisen from fusion of an Orf DNA binding domain to a His-Me finger nuclease domain ( 83 ). Our analyzes of genomic neighborhood support this hypothesis; the corresponding genes are potentially co-transcribed with genes encoding proteins from the SSB (PF00436) and DnaB_2 (PF07261) Pfam families.…”

Bacteriophage lambda: Early pioneer and still relevant

“…Lambda also carries two genes in its nin region that affect recombination, orf/ ninB and rap/ninG. Orf protein is a recombination mediator that binds to host single-strand binding protein and ssDNA (Maxwell et al , 2005; Curtis et al , 2011 and 2014), and Rap protein is a DNA structure-specific endonuclease that cleaves Holliday junction branch points (Sharples et al , 1998; Poteete et al , 2002). Homologous recombination occurs by several different pathways, and these phage-encoded proteins participate with host proteins in various ways (see for example Poteete, 2013).…”

“…Near the 3'-end of lambda's early right operon lie ten contiguous nonessential nin genes, so named because they are absent in large deletions that remove tR2 and make transcription of the essential gene Q (and thus the late genes) high enough to allow the phage to be N independent (Court and Sato, 1969). The nin genes include the ren, orf (ninB), rap (ninG) and orf221 , genes that encode protection from rexAB exclusion (Toothman and Herskowitz, 1980b), a homologous recombination function (Tarkowski et al , 2002; Curtis et al , 2014), a Holliday junction resolvase active in homologous recombination (Barik, 1993; Sharples et al , 2004) and a serine/threonine/tyrosine protein phosphatase (Voegtli et al , 2000) whose role is unknown, respectively.…”