Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Novel Nucleic Acid Binding Properties of Human UVSSA: A Component Protein of Transcription Coupled DNA Repair Complex (#302)

Hiral Mistry 1 , Gagan Deep Gupta 1
  1. Bhabha Atomic Research Centre, Trombay, MAHARASHTRA, India

UV stimulated scaffold protein A (UVSSA) is a newly identified 709 residues long protein of the intricate Transcription Coupled Nucleotide Excision Repair Complex (TCR/TC-NER)- a dedicated and specialized repair pathway dealing with DNA lesions on the actively transcribing genes1–3. Efficient interplay of UVSSA with other TCR component proteins is critical for transcription-repair coupling and successful DNA repair 4,5. Here, we illustrate the bioinformatics, biophysical and biochemical characterization of human UVSSA protein through its expression and purification in the bacterial expression system. This is the first ever study reporting purification of any homolog of UVSSA protein and the first ever characterization of the protein for nucleic acid binding activity. A combination of bioinformatics and biophysical techniques demonstrated the protein to be intrinsically disordered with two major domains, N-terminal VHS domain (1-150aa) and C-terminal DUF2043 domain (495-605aa). We have generated several N and C terminal partial UVSSA protein constructs and both the domain constructs co-purifies huge quantities of nucleic acid particularly RNA during Ni- based affinity chromatography. CD spectra of both the domain constructs displayed characteristic negative peaks of alpha helical proteins complementing the results obtained by PSIPred 4.0 software. The purified proteins were analysed for their appropriate folding, monodispersive nature and homogeneity and molecular mass using 1H NMR spectra, DLS and Gel filtration methods. The RNA binding activity of the UVSSA domain constructs was explored using electrophoretic mobility shift assay (EMSA) and biophysical techniques. The experiments revealed that the C-terminal region beyond the DUF domain exhibits robust binding to RNA as well as to DNA as compared to the VHS domain, whereas the DUF domain alone does not show any RNA binding activity. The identification of novel nucleic acid binding activity associated with UVSSA may help reveal other incognito functions and a deeper insight of the TCR pathway.

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