Xeroderma pigmentosum A homolog from Hydra partially complements DNA repair defect in human XPA-deficient cells.

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.

DNA Repair Repertoire of the Enigmatic Hydra.

Since its discovery by Abraham Trembley in 1744, hydra has been a popular research organism. Features like spectacular regeneration capacity, peculiar tissue dynamics, continuous pattern formation, unique evolutionary position, and an apparent lack of organismal senescence make hydra an intriguing animal to study. While a large body of work has taken place, particularly in the domain of evolutionary developmental biology of hydra, in recent years, the focus has shifted to molecular mechanisms underlying various phenomena. DNA repair is a fundamental cellular process that helps to maintain integrity of the genome through multiple repair pathways found across taxa, from archaea to higher animals. DNA repair capacity and senescence are known to be closely associated, with mutations in several repair pathways leading to premature ageing phenotypes. Analysis of DNA repair in an animal like hydra could offer clues into several aspects including hydra's purported lack of organismal ageing, evolution of DNA repair systems in metazoa, and alternative functions of repair proteins. We review here the different DNA repair mechanisms known so far in hydra. Hydra genes from various DNA repair pathways show very high similarity with their vertebrate orthologues, indicating conservation at the level of sequence, structure, and function. Notably, most hydra repair genes are more similar to deuterostome counterparts than to common model invertebrates, hinting at ancient evolutionary origins of repair pathways and further highlighting the relevance of organisms like hydra as model systems. It appears that hydra has the full repertoire of DNA repair pathways, which are employed in stress as well as normal physiological conditions and may have a link with its observed lack of senescence. The close correspondence of hydra repair genes with higher vertebrates further demonstrates the need for deeper studies of various repair components, their interconnections, and functions in this early metazoan.

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

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 (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. 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.