UvrB protein of Corynebacterium pseudotuberculosis complements the phenotype of knockout Escherichia coli and recognizes DNA damage caused by UV radiation but not 8-oxoguanine in vitro

• Published in: Gene 2018-01, Vol.639, p.34-43
• Main Authors: Catarina Teodoro Castro, Bárbara, Cançado de Faria, Rafael, Faria, Bruna Franciele, Azevedo, Vasco, Lara dos Santos, Luciana, Júnior, Moacyr Comar, Machado, Carlos Renato, de Oliveira Lopes, Débora
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bioinformatics; Cloning, Molecular; Corynebacterium pseudotuberculosis; Corynebacterium pseudotuberculosis - genetics; Corynebacterium pseudotuberculosis - metabolism; Corynebacterium pseudotuberculosis - radiation effects; DNA Damage; DNA repair; Escherichia coli - genetics; Functional complementation; Gene Knockdown Techniques; Guanine - analogs & derivatives; Guanine - metabolism; NER; Sequence Homology, Amino Acid; Ultraviolet Rays; UvrB
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2017.09.068

In prokaryotic cells, the UvrB protein plays a central role in nucleotide excision repair, which is involved in the recognition of bulky DNA lesions generated by chemical or physical agents. The present investigation aimed to characterize the uvrB gene of Corynebacterium pseudotuberculosis (CpuvrB) and evaluate its involvement in the DNA repair system of this pathogenic organism. In computational analysis, the alignment of the UvrB protein sequences of Escherichia coli, Mycobacterium tuberculosis, Bacillus caldotenax and Corynebacterium pseudotuberculosis showed high similarity and the catalytic amino acid residues and functional domains are preserved. A CpUvrB model was constructed by comparative modeling and presented structural similarity with the UvrB of E. coli. Moreover, in molecular docking analysis CpUvrB showed favorable interaction with EcUvrA and revealed a preserved ATP incorporation site. Heterologous functional complementation assays using E. coli uvrB-deficient cells exposed to UV irradiation showed that the CpUvrB protein contributed to an increased survival rate in relation to those in the absence of CpUvrB. Damaged oligonucleotides containing thymine dimer or 8-oxoguanine lesion were synthesized and incubated with CpUvrB protein, which was able to recognize and excise UV irradiation damage but not 8-oxoguanine. These results suggest that CpUvrB is involved in repairing lesions derived from UV light and encodes a protein orthologous to EcUvrB. •CpUvrB protein complements uvrB-deficient E. coli phenotype under UV irradiation.•In in vitro assay CpUvrB is involved in repair of thymine dimers but not recognized 8-oxoguanine lesion.•The protein CpUvrB presents functional domains and catalytic amino acids residues preserved.•CpUvrB binds to ATP and EcUvrA in in silico assays.