Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins

Abstract

Similarity between the DNA substrates and products of integrase-mediated site-specific recombination reactions results in a single recombinase enzyme being able to catalyze both the integration and excision reactions. The control of directionality in these reactions is achieved through a class of small accessory factors that favor one reaction while interfering with the other. These proteins, which we will refer to collectively as recombination directionality factors (RDFs), play architectural roles in reactions catalyzed by their cognate recombinases and have been identified in conjunction with both tyrosine and serine integrases. Previously identified RDFs are typically small, basic and have diverse amino acid sequences. A subset of RDFs, the cox genes, also function as transcriptional regulators. We present here a compilation of all the known RDF proteins as well as those identified through database mining that we predict to be involved in conferring recombination directionality. Analysis of this group of proteins shows that they can be grouped into distinct sub-groups based on their sequence similarities and that they are likely to have arisen from several independent evolutionary lineages. This compilation will prove useful in recognizing new proteins that confer directionality upon site-specific recombination reactions encoded by plasmids, transposons, phages and prophages.

Figure 1

Phenogram of RDFs. A tree based on degrees of similarities between RDFs was calculated with CLUSTALX (using the default parameters from http://web.tiscalinet.it/biologia/) and rendered with the DrawGram program (from the PHYLIP package at http://evolution.genetics.washington.edu). The vertical bars indicate groups of RDFs that stay together during multiple cycles of tree generation. The groups are named (as shown on the right) according to a member for which there is experimental evidence of RDF activity.

Figure 2

Sequence alignments of RDFs. RDFs within individual families are shown using alignments derived from CLUSTALX analysis (using the default parameters as in Fig. 1). Amino acid residues that are identical in 65% of the sequences are highlighted in red and residues that are similar among at least 75% of sequences are shown in blue. Similarity groupings were based on positive scoring substitutions as determined by the BLOSUM 85 substitution matrix (33). The location of a putative helix–turn–helix DNA binding motif is shown above the L5–SAM–SLP1 and P22 families. In cases where RDFs from different sources are of identical sequence, only one was used in the alignment and the view of each alignment is limited to a 150 residue segment containing the related sequences.

Figure 3

Distribution of isoelectric focusing point (pI) values among RDFs. The predicted pI was calculated for each RDF using Compute pI/MW (http://www.expasy.ch). The number of RDFs with pIs within 0.5 pH intervals was determined and plotted. The majority of the proteins are basic, with only 10 of the 63 RDFs having a pI < 7. Five of the seven proteins (Lambda, HK97, HK022, 434, H19J) in the 11–11.5 range have identical sequences.