Analysis of the conserved NER helicases (XPB and XPD) and UV-induced DNA damage in Hydra

• Published in: Biochimica et Biophysica Acta (BBA) - General Subjects Vol. 1862; no. 9; pp. 2031 - 2042
• Main Authors: Galande, Alisha A., Perween, Nusrat, Saijo, Masafumi, Ghaskadbi, Saroj S., Ghaskadbi, Surendra
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2018; Elsevier BV
• Subjects: Amino Acid Sequence; Animals; cell lines; Cells, Cultured; Cyclobutane pyrimidine dimers; DNA; DNA Damage; DNA Damage - radiation effects; DNA Repair; DNA Repair - radiation effects; enzyme activity; gene expression; Gene Expression Regulation; Gene Expression Regulation - radiation effects; genome; germplasm conservation; Humans; Hydra; Hydra - enzymology; Hydra - genetics; Hydra - radiation effects; in situ hybridization; Nucleotide excision repair; photosensitivity disorders; Phylogeny; precipitin tests; proteins; quantitative polymerase chain reaction; reverse transcriptase polymerase chain reaction; Sequence Homology; thymine; Ultraviolet light; Ultraviolet Rays; Xeroderma Pigmentosum; Xeroderma Pigmentosum - genetics; Xeroderma Pigmentosum - metabolism; Xeroderma Pigmentosum Group D Protein; Xeroderma Pigmentosum Group D Protein - genetics; Xeroderma Pigmentosum Group D Protein - metabolism; XPB; XPD; A; TC-NER; C; CPDs; UniProt; RNA; TTD; G; kDa; XPB; XPD; bp; 6-4PPs; PDB; Ultraviolet light; NLS; T; MEGA; NCBI; cDNA; BLAST; UV; GG-NER; Nucleotide excision repair; Hydra; BER; CS; DNA; TFIIH; XP; NER; Cyclobutane pyrimidine dimers
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2018.06.017

Nucleotide excision repair (NER) pathway is an evolutionarily conserved mechanism of genome maintenance. It detects and repairs distortions in DNA double helix. Xeroderma Pigmentosum group B (XPB) and group D (XPD) are important helicases in NER and are also critical subunits of TFIIH complex. We have studied XPB and XPD for the first time from the basal metazoan Hydra which exhibits lack of organismal senescence. In silico analysis of proteins was performed using MEGA 6.0, Clustal Omega, Swiss Model, etc. Gene expression was studied by in situ hybridization and qRT-PCR. Repair of CPDs was studied by DNA blot assay. Interactions between proteins were determined by co- immunoprecipitation. HyXPB and HyXPD were cloned in pET28b, overexpressed and helicase activity of purified proteins was checked. In silico analysis revealed presence of seven classical helicase motifs in HyXPB and HyXPD. Both proteins revealed polarity-dependent helicase activity. Hydra repairs most of the thymine dimers induced by UVC (500 J/m2) by 72 h post-UV exposure. HyXPB and HyXPD transcripts, localized all over the body column, remained unaltered post-UV exposure indicating their constitutive expression. In spite of high levels of sequence conservation, XPB and XPD failed to rescue defects in human XPB- and XPD-deficient cell lines. This was due to their inability to get incorporated into the TFIIH multiprotein complex. Present results along with our earlier work on DNA repair proteins in Hydra bring out the utility of Hydra as model system to study evolution of DNA repair mechanisms in metazoans. •Hydra XPB and XPD are homologs of human XPB and XPD proteins.•Phylogenetic analysis and homology modeling indicate close relationship to vertebrate XPB and XPD.•Both HyXPB and HyXPD exhibit polarity-dependent helicase activity.•Hydra is able to rectify CPDs caused due to UV exposure by 72 h post-radiation.•HyXPB and HyXPD were unable to integrate into human TFIIH and failed to complement human XPB- and XPD-deficient cell lines.