title: Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites.
abstract: Homologous recombination (HR) is a highly conserved pathway for the repair of chromosomes that harbor DNA double-stranded breaks (DSBs). The recombinase RAD51 plays a key role by catalyzing the pairing of homologous DNA molecules and the exchange of information between them. Two putative DMC1 homologs (DMC1A and DMC1B) have been identified in Giardia duodenalis. In terms of sequences, GdDMC1A and GdDMC1B bear all of the characteristic recombinase domains: DNA binding domains (helix-turn-helix motif, loops 1 and 2), an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis. Because GdDMC1B is expressed at the trophozoite stage and GdDMC1A is expressed in the cyst stage, we cloned the giardial dmc1B gene and expressed and purified its protein to determine its activities, including DNA binding, ATP hydrolysis, and DNA strand exchange. Our results revealed that it possessed these activities, and they were modulated by divalent metal ions in different manners. GdDMC1B expression at the protein and transcript levels, as well as its subcellular localization in trophozoites upon DNA damage, was assessed. We found a significant increase in GdDMC1B transcript and protein levels after ionizing radiation treatment. Additionally, GdDMC1B protein was mostly located in the nucleus of trophozoites after DNA damage. These results indicate that GdDMC1B is the recombinase responsible for DSBs repair in the trophozoite; therefore, a functional Rad51 role is proposed for GdDMC1B.
authors.affiliation: Genetics and Molecular Biology Department, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional No. 2508, C.P. 07360, México D.F., Mexico.
authors.forename: Ana Laura
authors.initials: AL
authors.lastname: Torres-Huerta
authors.affiliation: Genetics and Molecular Biology Department, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional No. 2508, C.P. 07360, México D.F., Mexico.
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Martínez-Miguel
authors.affiliation: Genetics and Molecular Biology Department, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional No. 2508, C.P. 07360, México D.F., Mexico.
authors.forename: María Luisa
authors.initials: ML
authors.lastname: Bazán-Tejeda
authors.affiliation: Genetics and Molecular Biology Department, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional No. 2508, C.P. 07360, México D.F., Mexico. Electronic address: roberm@cinvestav.mx.
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Bermúdez-Cruz
mesh_terms: D000595:Amino Acid Sequence; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D009698:Nucleoproteins / Q000378:metabolism; D000072417:Protein Domains; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053058:Trophozoites / Q000201:enzymology* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D009698:Nucleoproteins; D051135:Rad51 Recombinase
keywords: DNA double strand breaks; DNA repair; Gamma radiation; Giardia duodenalis; Homologous recombination; RAD51