Organisms with linear chromosomes face two dilemmas. Their chromosome ends have to be protected from degradation and fusion and, in addition, chromosome ends require a specialized replication strategy because conventional DNA polymerases are unable to complete the 5'end of a new DNA strand. Eukaryotes solve these problems by capping linear chromosomes with a specialized nucleoprotein complex, the telomere. The DNA of vertebrate telomeres is composed of a highly conserved tandemly repeated hexamer with the sequence TTAGGG. Based on our preliminary studies and by analogy to telomeres in unicellular organisms, we expect that the TTAGGG repeats interact with specific telomere proteins to form a nucleoprotein complex that protects the chromosome end. Our objective is to describe the protein components of this complex and to understand their role in telomere function. We have identified two candidate telomere proteins in vertebrate cells and propose to clone their cDNAs. The cDNAs will be used to generate recombinant proteins and antisera. Verification of the identity of the cloned genes will be based on biochemical and antigenic similarity of recombinant factors and the putative telomere proteins in extracts. Once the identity of the cloned genes is established, we will use immuno- cytochemical techniques to identify the chromosomal position of these putative telomere factors. If the factors are associated with chromosome ends and/or show amino acid sequence similarity to telomere proteins in unicellular organisms, we will proceed with their functional analysis. One part of the functional studies will focus on the molecular interactions of telomere factors with telomeric DNA and with telomerase. We will examine the nature of the telomeric DNA-protein complex and determine whether DNA ends in this complex are protected. Furthermore, we will explore the possibility that telomerase is regulated by telomere proteins. In addition, the functional studies will ask whether loss of the telomere proteins leads to telomere malfunction in mammalian cells. We will analyze telomere proteins in cells whose telomeres show signs of malfunction, such as cells with decaying telomeres and cells with chromosome end associations. Secondly, we will evaluate the molecular and cytological effects of null mutations in genes for telomere proteins in tissue culture cells. The proposed experiments are designed to reveal the structure and mechanism of human telomeres. A better understanding of the molecular cytology of human telomeres is important in view of the possibility that telomere malfunction may contribute to genome instability in transformed and senescent cells.
Showing the most recent 10 out of 22 publications
