Novel G-DNA binding proteins from the ciliate Tetrahymena thermophila: purification, characterization, cloning and functional analyses

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Lu, Quan
Major Professor
Eric Henderson
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G-DNA is a family of novel four-stranded DNA structures characterized by motifs called G-quartets. Evidence is growing to suggest that G-DNA exists and plays biological roles in vivo. In order to further elucidate the functions of G-DNA, we have studied proteins that specifically bind to the DNA structure;Two G-DNA binding activities, TGPI and TGP3, were purified from the ciliate Tetrahymena thermophila. Based on the peptide sequences obtained from direct internal peptide sequencing, the cDNAs coding for the genes were cloned. Deduced protein sequences showed that TGP1 and TGP3 are novel proteins but share significant homology with each other. Furthermore, the two proteins contain an intriguing sequence pattern with two repetitive and homologous motifs flanking an extensively hydrophilic region. We suggest that this shared sequence pattern may represent a novel G-DNA binding motif,;To address the biological functions of these two novel G-DNA binding proteins, we have employed a newly-developed gene knock out technique and generated Tetrahymena strains with each of the two genes completely disrupted in the macronucleus. Both knock-out strains (TGPlKO and TGP3KO) grow normally suggesting that neither of the genes is essential for cell growth and survival. Nevertheless, detailed nuclear staining analysis revealed micronuclear aberrance characterized by higher occurrence of multiple micronuclei in both knock-out cells, suggesting a faulty control of the micronuclear division. More interestingly, TGPlKO cells showed an increased TGP3 activity, implying that these two proteins may share some aspects of biological functions. In addition to the gene knock-out experiments, we also did nuclear fractionation experiment, demonstrating that both TGPI and TGP3 localize mainly in the nuclei. Based on these data, we propose a model, in which TGP proteins coordinate to function in micronuclear division through binding to the G-DNA structure formed between telomeres of two sister chromatids.

Fri Jan 01 00:00:00 UTC 1999