The overall goal of this project is to learn about telomere structure and function by determining the x-ray crystal structures of nucleoprotein complexes that compose telomeres. Currently, our efforts ar focused on two cloned and characterized proteins that are known to be components of telomeres in vivo: (i) the telomere binding protein from Oxytricha nova which interacts with single-strand DNA at the very ends of telomeres and (ii) RAP1 from yeast which interacts with the double-stranded DNA region of telomeres and has been shown to participate in regulating telomere length. The telomere binding protein from Oxytricha nova interacts with single- strand DNA (sequence TTTTGGGGTTTTGGGG) that occurs at the very ends of telomeres. The telomere binding protein consists of two subunits; a 56 Kd alpha-subunit that interacts single-strand (TTTTGGGG)n DNA in a sequence specific manner and a 41 KD beta-subunit that alters the DNA in a sequence specific manner and a 41 KD beta-subunit that alters the DNA binding properties of the alpha-subunit. The alpha- and beta-subunits do not interact in solution in the absence of DNA, but together with (TTTTGGGG)n DNA they form a very tight ternary complex containing the alpha-subunit, the beta-subunit, and DNA. We have obtained crystals of (i) the alpha-subunit complexed with TTTTGGGG DNA that diffract to 2.7A resolution, (ii) the beta-subunit in th presence of GGGG DNA, and (iii) the entire ternary complex containing the alpha-subunit, the beta- subunit, and GGGGTTTTGGGG DNA that diffract to at least 3 A resolution. We are now poised for a very detailed look at the telomere binding protein-DNA complex which will greatly enhance our understanding of how the two subunits of this protein interact with DNA and with each other to form the nucleoprotein complex that caps the ends of chromosomes. RAP1 is associated with yeast telomeres in vivo and is necessary for proper regulation of telomere length. Mutations in this protein give rise to either elongated or shortened telomeres. RAP1 is a sequence- specific double-stranded DNA binding protein that interacts with the sequence ACACCCACACACC which occurs every 30-40 base pairs in yeast telomeres. Interestingly, RAP1 has been shown to bend DNA such that it may give rise to a higher order structure in yeast telomeres. We have begun efforts to obtain large quantities of RAP1 for crystallization and structure determination. That improper maintenance of telomere length and structure limits a cells ability to divide and results in senescence has been clearly established. Indeed, it is proposed that telomeres play a critical role in cell division and in the """"""""senescence program: """""""" in higher eukaryotes. Understanding the structure of the nucleoprotein complexes that compose telomeres will lead to a better overall understanding of the structure and function of telomeres.
| Froelich-Ammon, S J; Dickinson, B A; Bevilacqua, J M et al. (1998) Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha. Genes Dev 12:1504-14 |
