Biochemical characterization of clinically relevant mutations of human Translin

DNA damage in all living cells is repaired with very high efficiency and nucleic acid binding proteins play crucial roles in repair associated processes. Translin is one such evolutionarily conserved nucleic acid interacting protein speculated to be a part of the DNA repair protein network. It is also involved in activation of RNA-induced silencing complex (RISC) along with Translin-associated factor X (TRAX) as the C3PO (component 3 promoter of RISC) complex. In the present work, we characterized ten clinically relevant variants of the human Translin protein using bioinformatic, biochemical, and biophysical tools. Bioinformatic studies using DynaMut revealed 9 out of the 10 selected mutations the Translin protein. Further analysis revealed that some mutations lead to changes in interactions with neighbouring residues in the protein structure. Using site directed mutagenesis, the point substitution variants were generated, corresponding proteins were overexpressed and purified using Ni-NTA affinity chromatography. Purified proteins form octamers similar to wild type (WT) Translin, as observed using native polyacrylamide gel electrophoresis (PAGE), gel filtration, and dynamic light-scattering (DLS) analysis. These octamers are functional and bind to single-stranded DNA (ssDNA) as well as single-stranded RNA (ssRNA) substrates. The mutant Translin proteins interact with wild type TRAX and form corresponding C3PO complexes. The C3PO complexes formed by all Translin variants with TRAX are functional in-vitro and show endoribonuclease activity. However, significant differences were observed in the extent of RNase activity in vitro. In conclusion, the clinically relevant mutations in Translin protein analysed by us exert their effect by modulating the RNase activity of the protein without altering its DNA-dependant function.

Automated summary

In this study, we characterized ten clinically relevant variants of human Translin protein and found that these variants can modulate the RNase activity of the protein without altering its DNA-dependent function.