In silico data analyses of recombinases GdDMC1A and GdDMC1B from Giardia duodenalis

Giardia duodenalis is a worldwide protozoa known causing diarrhea in all vertebrates, humans among these. Homologous recombination is a mechanism that provides genomic stability. Two putative recombinases were identified in G. duodenalis genome: GdDMC1A and GdDMC1B. In this article, we describe the identification of conserved domains in GdDMC1A and GdDMC1B, such as: DNA binding domains (Helix-turn-helix motif, loops 1 and 2) and an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis, phylogenetic analyses among assemblages and three-dimensional structure modeling of these recombinases using bioinformatics tools. Also, experimental data is described about LD50 determination for ionizing radiation in trophozoites of G. duodenalis. Additionally, as recombinases, GdDMC1A and GdDMC1B were used to rescue a defective Saccharomyces cerevisiae Δ rad51 strain under genotoxic conditions and data is described. The data described here are related to the research article entitled “Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites” (Torres-Huerta et al.,) [1].

In silico analyses DMC1B and DMC1A 3D structure prediction DL50 for ionizing radiation a b s t r a c t Giardia duodenalis is a worldwide protozoa known causing diarrhea in all vertebrates, humans among these. Homologous recombination is a mechanism that provides genomic stability. Two putative recombinases were identified in G. duodenalis genome: GdDMC1A and GdDMC1B. In this article, we describe the identification of conserved domains in GdDMC1A and GdDMC1B, such as: DNA binding domains (Helix-turn-helix motif, loops 1 and 2) and an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis, phylogenetic analyses among assemblages and three-dimensional structure modeling of these recombinases using bioinformatics tools. Also, experimental data is described about LD50 determination for ionizing radiation in trophozoites of G. duodenalis. Additionally, as recombinases, GdDMC1A and GdDMC1B were used to rescue a defective Saccharomyces cerevisiae Δ rad51 strain under genotoxic conditions and data is described.
The data described here are related to the research article entitled "Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites" (Torres-Huerta et al.,) [1]. &

Value of the data
Bioinformatics tools allow to identify conserved domains and/or function in recombinases GdDMC1A and GdDMC1B.
Structure-based protein alignment allows to identify conserved domains/functions in recombinases GdDMC1A and GdDMC1B with respect to other recombinases.
Phylogenetic analyses allow to cluster these recombinases with other organisms to determine whether they are closely related.
Comparative modeling analysis between predicted structure of GdDMC1A and GdDMC1B can identify differences in protein structure and make some predictions.

Data
Recombinases GdDMC1A and GdDMC1B amino acid sequences were compared with recombinases from other organisms (Structure-based sequence alignments) and phylogenetic reconstructions and 3D modeling were made to identify similarity (Figs. 1-3). Also, GdDMC1A and GdDMC1B protein sequences from other assemblages within Giardia duodenalis were identified Fig. 2. Evolutionary relationships of GdDMC1A and GdDMC1B. A phylogenetic tree was inferred using the Neighbor-Joining method [2]. The optimal tree with the sum of branch length ¼ 2.30818981 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [3] and are in the units of the number of amino acid substitutions per site. The analysis involved 13 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 300 positions in the final dataset. Evolutionary analyses were conducted as described previously [4].   Table 1) and compared among them (Figs. 4 and 5). Finally, GdDMC1A and GdDMC1B were expressed in a rad51 defective yeast strain (Fig. 6) and LD50 using ionizing radiation was calculated in G. duodenalis trophozoites (Fig. 7).

Experimental design, materials and methods
Bioinformatic tools (Structure-based sequence alignments and phylogenetic reconstructions) were used to create Figs. 1-5. Fig. 6 shows transforming a rad51 defective Saccharomyces cerevisiae strain with either GdDMC1A or GdDMC1B containing plasmids looking for a rescue phenotype under DNA damaging conditions and Fig. 7 is generated by determining LD50 at 12, 32 y 48 h after ionizing radiation exposure in G. duodenalis. This experimental design was used only for this work. Table 1 Data of recombinases from different assemblages of Giardia duodenalis. In the sequence name the first letter (A, A2, B, E) indicates the assemblage, after the hyphen the isolate of Giardia is shown and the last number 1 or 2 corresponds to proteins GdDMC1A or GdDMC1B respectively.  . 4. Alignment of GdDMC1A and GdDMC1B (indicated in the name for each sequence as 1 or 2 respectively) from different assemblages of Giardia duodenalis based on secondary structure of ScRad51 (1SZP). Sequences used are described in Table 1.
Colors are as indicated in Fig. 1. This Structure-based protein sequence alignment was performed with ESPript program [5].  5. Evolutionary relationships of GdDMC1A and GdDMC1B among assemblages of Giardia duodenalis. The evolutionary history was inferred using the Neighbor-Joining method [2]. The optimal tree with the sum of branch length¼ 0.52279652 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [3] and are in the units of the number of amino acid substitutions per site. The analysis involved 10 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 348 positions in the final dataset. Evolutionary analyses were conducted as previously described [4]. As in Table 1, 1 refers to A and 2 refers to B. Fig. 6. rGdDMC1A and rGdDMC1B do not complement a rad51 defective Saccharomyces cerevisiae. GdDMC1A and GdDMC1B were cloned in pRS316 vector, then they were transfected into a Δrad51 Saccharomyces cerevisiae. The ability to rescue Rad51 mutation in transfected yeast was evaluated. DNA was damaged with genotoxic agents (25 mM hydroxyurea and 50 J/m 2 UV light). Serial dilutions were spotted into YPD plates containing the genotoxic agent and yeast growth was observed. A wild type (WT, 3595) strain was used to compare with its corresponding Δrad51 strain and the transfected yeasts with Giardial recombinases. As a positive control ScRad51 was used to complement Δrad51.