Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica.
BACKGROUND
In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. RAD52 epistasis group genes involved in recombinational DNA repair, including mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 and rad59 genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in Entamoeba histolytica the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.


RESULTS
In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote E. histolytica using UV-C (150 J/m2) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In E. histolytica genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the E. histolytica RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear foci-like structures in E. histolytica trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands in vitro.


CONCLUSIONS
E. histolytica genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear foci-like structures. Functional assays confirmed that EhRAD51 is a bonafide recombinase. These data provided the first insights about the potential roles of the E. histolytica RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.
López-Casamichana
Mavil
M
Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, México DF, México. gerry2410@yahoo.com
Orozco
Esther
E
Marchat
Laurence A
LA
López-Camarillo
César
C
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded; D053938:DNA Fragmentation; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D020287:In Situ Nick-End Labeling; D051135:Rad51 Recombinase / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014466:Ultraviolet Rays
D017931:DNA Primers; D051135:Rad51 Recombinase