title: Identification of nucleic acid binding sites on translin-associated factor X (TRAX) protein.
abstract: Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.
authors.affiliation: High Pressure & Synchrotron Radiation Physics Division, Bhabha Atomic Research Centre, Mumbai, India.
authors.forename: Gagan Deep
authors.initials: GD
authors.lastname: Gupta
authors.forename: Vinay
authors.initials: V
authors.lastname: Kumar
mesh_terms: D001665:Binding Sites / Q000235:genetics*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009696:Nucleic Acids / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009696:Nucleic Acids; C095563:TSN protein, human; C498305:TSNAX protein, human