G4-DNA is a highly stable alternative DNA structure that can form spontaneously in guanine-rich regions of single-stranded DNA under physiological conditions. Since a number of biological processes create such single-stranded regions, G4-DNA occurrence must be regulated. To date, resolution of tetramolecular G4-DNA into single strands (G4-resolvase activity) has been observed only in recombinant RecQ DNA helicases. We previously reported that human cell lysates possess tetramolecular G4-DNA resolving activity (Harrington, C., Lan, Y., and Akman, S. (1997) J. Biol Chem. 272, 24631-24636). Here we report the first complete purification of a major non-RecQ, NTP-dependent G4-DNA resolving enzyme from human cell lysates. This enzyme is identified as the DEXH helicase product of gene DHX36 (also known as RHAU). G4-DNA resolving activity was captured from HeLa cell lysates on G4-DNA affinity beads and further purified by gel filtration chromatography. The DHX36 gene product was identified by mass spectrometric sequencing of a tryptic digest from the protein band on SDS-PAGE associated with activity. DHX36 was cloned within a His 6 -tagging vector, expressed, and purified from Escherichia coli. Inhibition and substrate resolution assays showed that recombinant DHX36 protein displayed robust, highly specific G4-DNA resolving activity. Immunodepletion of HeLa lysates by a monoclonal antibody to the DHX36 product removed ca. 77% of the enzyme from lysates and reduced G4-DNA resolving activity to 46.0 ؎ 0.4% of control, demonstrating that DHX36 protein is responsible for the majority of tetramolecular G4-DNA resolvase activity.G4-DNA is an alternative highly stable DNA structure forming within runs of guanine bases. It has been amply described previously (1). G4-DNA structures have the potential to disrupt normal duplex DNA; therefore, it might be expected that the genome would have minimized the usage of runs of deoxyguanosine. On the contrary, a growing body of data support the hypothesis that formation of G4-DNA in vivo is a recognized structural motif of specialized utility for key biological processes. Recent studies with a fluorescent G4-DNAbinding ligand, as well as a specific G4-DNA-binding protein, support the presence of G4-DNA structures located at human telomeres in vivo (2, 3). In addition to the aforementioned telomeres, other guanine-rich regions in human DNA readily form G4-DNA structures in vitro and make up specific genetic control elements, including the immunoglobin heavy chain switch region (4), guanine-rich regions of ribosomal DNA (5), the d(pCGG) repeats of the fragile X mental retardation gene (6), promoters of proliferation-associated genes, such as the c-MYC (7, 8), PDGF-A (9), RET (10), and the diabetes susceptibility locus IDDM2 promoter (11). It has been shown that a unimolecular G4-DNA structure has a repressor function in the c-MYC promoter (8). Compounds that stabilize G4-DNA in vivo have generated much interest because of their antitumor activity, suggesting that G4-DNA structures might be ...

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Although the decision between stem cell self-renewal and differentiation has been linked to cell-cycle modifications, our understanding of cell-cycle regulation in stem cells is very limited. Here, we report that FBF/Pumilio, a conserved RNA-binding protein, promotes self-renewal of germline stem cells by repressing CKI-2 Cip/Kip , a Cyclin E/Cdk2 inhibitor. We have previously shown that repression of CYE-1 (Cyclin E) by another RNA-binding protein, GLD-1/Quaking, promotes germ cell differentiation. Together, these findings suggest that a post-transcriptional regulatory circuit involving FBF and GLD-1 controls the self-renewal versus differentiation decision in the germline by promoting high CYE-1/CDK-2 activity in stem cells, and inhibiting CYE-1/CDK-2 activity in differentiating cells.

Appropriately regulated gene expression requires a suitable promoter. A number of promoters have been isolated and shown to be functional in plants, but only a few of them activate transcription of transgenes at high levels constitutively. We report here the cloning and characterization of a novel, constitutively expressed promoter isolated from Cestrum yellow leaf curling virus (CmYLCV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family. The CmYLCV promoter is highly active in callus, meristems and vegetative and reproductive tissues in Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Zea mays and Oryza sativa. Furthermore, the level of expression is comparable to, or higher than, that from the CaMV 35S, the 'super-promoter' or the maize ubiquitin 1 promoters, three frequently used promoters in agricultural biotechnology. The heritable, strong and constitutive activity in both monocotyledonous and dicotyledonous plants, combined with the extremely narrow CmYLCV host range, makes the CmYLCV promoter an attractive tool for regulating transgene expression in a wide variety of plant species.