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. 2010 Jul;38(12):3952-62.
doi: 10.1093/nar/gkq096. Epub 2010 Mar 1.

Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs

Anne Lopes  1 , Jihane Amarir-Bouhram, Guilhem Faure, Marie-Agnès Petit, Raphaël Guerois
Affiliations

Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs

Anne Lopes et al. Nucleic Acids Res. 2010 Jul.

Abstract

Homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. No less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (UvsX and Gp2.5) from virulent, and four (Sak, Red beta, Erf and Sak4) from temperate phages. Using profile-profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. We show that Sak, Red beta and Erf belong to a common large superfamily adopting a shortcut Rad52-like fold. Remote homologs of Sak4 are predicted to adopt a shortcut Rad51/RecA fold and are discovered widespread among phage genomes. Unexpectedly, within temperate phages, gene-context analyses also pinpointed the presence of distant Gp2.5 homologs, believed to be restricted to virulent phages. All in all, three major superfamilies of phage recombinases emerged either related to Rad52-like, Rad51-like or Gp2.5-like proteins. For two newly detected recombinases belonging to the Sak4 and Gp2.5 families, we provide experimental evidence of their recombination activity in vivo. Temperate versus virulent lifestyle together with the importance of genome mosaicism is discussed in the light of these novel recombinases. Screening for these recombinases in genomes can be performed at http://biodev.extra.cea.fr/virfam.

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Figures

Figure 1.
Figure 1.
Homology relationships between recombinases revealed by iterative profile–profile comparisons. Network of homology relationships detected between the four recombinases superfamilies Redβ/RecT (magenta), Erf (orange), Sak (red) and Sak4 (purple). Nodes represent the bacteriophage genomes for which a homologous recombinase was detected. Edges connect recombinases which were found homologous from the profile–profile comparison using HHsearch. Bold coloured edges correspond to significant hit obtained from the initial screening of the bacteriophage 28 300 profiles database with the four recombinase profiles. A second iteration using the detected recombinase profiles as queries revealed additional relationships among the same superfamily (light grey edges) and, most importantly, between different superfamilies (black edges). Nodes with a black outline pinpoint novel recombinases annotated as unknown function so far. Graph created using the Osprey program (53).
Figure 2.
Figure 2.
Structural models of the recombinases found evolutionarily related to the Rad52 superfamily. (A) Optimal sequence alignment resulting from the structural modelling procedure between the sequences of human Rad52 corresponding to the PDB (1H2I), Sak (from phage ul36), Erf (D3), Redβ (933W). Secondary structures are indicated on top of the alignment from blue to red colour as in (B and C). Truncated secondary structures between Rad52 and the phage recombinases are indicated by dashed secondary structures. The red star indicates the position of K152 in Rad52 whose mutation abrogated single-strand binding. (B) Cartoon representation of the Rad52 crystallized domain and of the Redβ structural model with the red star pinpointing the side-chain of K152 and R161, respectively. (C) Surface representation of Rad52 11-mer oligomeric form as observed in the crystal structure 1H2I together with the 11-mer models of the three recombinases Sak, Erf and Redβ. On the left, the surface is coloured in either light cyan or orange to help visualizing the external and internal ring, respectively. On the right the surface is coloured with respect to the conservation index calculated using Rate4Site program, conservation grade increasing from white to red.
Figure 3.
Figure 3.
Structural Model of Sak4, a shortcut homolog of RecA/Rad51 superfamily. (A) Comparison between Sak4 from Lactococcus phage Φ31 (gb:AAC48871) and other members of the RecA/RadA/Rad51 superfamily, E. coli RecA (B7N6S9), Pyrococcus abyssi RadA (Q9V233) and RadB (Q9V2F6), human Rad51 (Q06609), Rad51B (O15315), Rad51C (O43502), Rad51D (O75771), XRCC2 (O43543) and XRCC3 (O43542). Secondary structures are indicated on top of the alignment from blue to red; pink and purple bars indicate the position of additional domains. (B) Ribbon representation of the structural model of Sak4 from Lactococcus phage Φ31 compared to that of RadB (pdb 2cvh), RecA (pdb 1u94) and Rad51 (pdb 1szp) X-ray structures.
Figure 4.
Figure 4.
Analysis of the gene context in the DNA replication and recombination module of several bacteriophages. (see also Supplementary Figure S5) (A) Phage names are indicated on top with the type of detected recombinase annotated below. ORFs are represented by ordered boxes with size related to the ORF length. Light and medium grey boxes indicate genes not considered in the analysis. The different predicted recombinases are colored as pink. Other colours, when identical, pinpoint genes sharing a homology relationship (either remote or close). Genes surrounding the homolog of 5′–3′ exonuclease RecE (yellow/red stripes) in P335 sensu lato (in fact, phage 4268), bIL309, TLS and ϕ12 phages. A Rad52 (pink box) or Rad51-like (dark pink box) recombinase is often found in a close neighbourhood of RecE, suggesting that in ϕ12 (the white gene labelled with a question mark) could itself be a recombinase. Some putative functions have been labelled by a short-term, ini, initiator; hel loader, DNA-C type helicase loader; ssb, single-strand binding; RecE, 5′–3′ exonuclease RecE; int, phage integrase; repr, repressor; DNA pol, DNA polymerase (Pol I-type); dut, dUTPase; endo, endonuclease; SF2 hel, superfamily 2 helicase; PriA, DNA primase. (B) Structural model of the Gp2.5-like protein identified in phage ϕ12. From left to right, ribbon representation of (i) the structure of Gp2.5 protein (phage T7) (PDB code: 1je5), (ii) the structural model of Gp2.5 distant homolog from phage ϕ12 and (iii) the structure of the single-strand binding (SSB) protein of E. coli (PDB code: 1eyg). Red stars in Gp2.5 structures pinpoint residues whose mutation abrogated single strand annealing activity [in (49) and in this study].
Figure 5.
Figure 5.
Single-strand annealing activities in vivo of the three superfamilies of recombinases. Efficiency of recombination is estimated by the ratio of rifampicin-resistant recombinants (generated by integration of the Maj32 single-strand oligonucleotide into rpoB), per viable cells. Each value corresponds to the average of at least three experiments, performed in an E. coli AB1157 derivative in which a given recombinase (x-axis) has been induced from a low-copy number pSC101 plasmid derivative. Spontaneous rifampicin-resistant mutants were obtained at a median frequency of 2 × 10−8. To ascertain the effect of each recombinase, as compared to the empty plasmid vector (‘No Recombinase’), a Student test was performed: double asterisks indicate significance at the 1% level, single asterisk indicate significance at the 5% level.
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