RESUMO
Nucleotide excision repair (NER) pathway is a DNA repair mechanism that rectifies a wide spectrum of DNA lesions. Xeroderma pigmentosum group of proteins (XPA through XPG) orchestrate the NER pathway in humans. We have earlier studied XPA homolog from Hydra (HyXPA) and found it to be similar to human XPA. Here, we examined if HyXPA can functionally complement human XPA-deficient cells and reduce their sensitivity to UV radiation. We found that HyXPA was able to partially rescue XPA-deficient human cells from UV by its binding to chromatin of UV-irradiated cells. However, HyXPA failed to bind replication protein A (RPA70), a key interacting partner of human XPA in NER pathway. This could be attributed to changes in certain amino acid residues that have occurred during evolution, leading to prevention of some interactions between Hydra and human proteins.
Assuntos
Cromatina/química , Reparo do DNA , DNA/genética , Evolução Molecular , Tolerância a Radiação/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Expressão Gênica , Teste de Complementação Genética , Humanos , Hydra , Plasmídeos/química , Plasmídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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 (500â¯J/m2) by 72â¯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. CONCLUSIONS: 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.