The integrity of telomeres is essential for chromosomal stability and, it has been found recently, other cellular functions including nuclear division. The replenishment of telomeric sequences, lost gradually as a result of conventional DNA replication, is carried out by the specialized ribonucleoprotein reverse transcriptase/telomerase, which adds telomeric DNA to a DNA 3' end by copying an internal RNA template. Recent data including kinetic studies show that the interaction of telomerase with primer sequences 5' to the 3' region involved in base pairing to the RNA template is functionally important in primer recognition and translocation during processive elongation. This interaction has been proposed to be carried out by a second (lagging product) binding site of telomerase in the current 'two-site' models. This proposal will dissect this interaction at the molecular level.
The specific aims are to 1) probe the specific contacts between the telomerase lagging product site and its DNA primer substrate by DNA footprinting analysis; 2) analyze the contribution of sequence-specific functional groups on DNA to the interaction of DNA primer with the lagging site of telomerase by functional modification interference assays; 3) study the role of the telomerase DNA in the interaction between telomerase and telomeric DNA primer. This study will provide insights into telomere biosynthesis and polymerization of nucleic acids in general.
