) Cellular mechanisms for maintaining and controlling telomere length constitute a critical arm of the regulatory network that enforces cell senescence and suppresses cell transformation and tumor outgrowth. Identification of the molecules and mechanisms essential to telomere maintenance is therefore central to an understanding of cell transformation. Telomeres in human somatic cells consist of 3-18 kb of duplex DNA containing reiterations of the TTAGGG repeat, and terminate with G-rich single stranded 3' overhangs 130-210 nt in length, which are referred to as G-tails. G-tails may provide sites for initiation of telomere replication by telomeres; protect telomeres from degradation or fusion; mediate interactions between telomeres during meiosis; direct chromosome attachment to sites in the nucleus; or regulate telomere length or telomerase activity. We have found that the ubiquitous and conserved mammalian protein, hnRNP D, binds tightly and specifically to telomeric G-tails and to G4 DNA structures formed by G-G pairing of G-rich telomeric repeats. We will study the interaction of hnRNP D with telomeric single-stranded G-tails and G4 DNA (Aim 1). We will study the function of hnRNP in vivo, by targeted mutation in ES cells and knockout mice (Aim 2). We will study regulation of hnRNP D and identify pathways in which it functions (Aim 3). We will identify a helicase active on telomeric G4 DNA structures (Aim 4). We will study the mechanism of telomerase-independent telomere maintenance in immortalized cells and tumor cells that lack telomerase understanding of mechanisms central to telomere maintenance in normal and transformed cells. These experiments will also have clear practical ramifications, because pathways of telomere maintenance that they identify will be potential targets for small molecule inhibitors of cell proliferation that can be used in treatment of malignancies.
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