Organization of mammalian telomere chromatin.

Abstract

Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes. They protect ends of chromosomes from fusion and degradation. The telomere DNA of most eukaryotes consists of stretches of simple repetitive DNA sequences with G and C rich strands. Telomeres from the many somatic cell lines are shortened with successive cell divisions. The shortening of telomeres is associated with aging and senescence and can serve as a molecular clock to prevent oncogenesis. Telomere length can be stabilized by telomerase, the reverse transcriptase that adds the telomeric repeats at the ends of chromosomes. Telomere nucleoprotein of low eukaryotes such as yeast is devoid of nucleosomes and organized as a special telomere-specific structure called the telosome, which is involved in telomere length regulation and protection. However, the organization of mammalian telomere nucleoprotein was not known. In this dissertation I have used biochemical and biophysical techniques to investigate the structure of mammalian telomere nucleoprotein. Techniques used include conventional nuclease probing of the structural features of telomere nucleoprotein as well as three novel techniques developed by me: (1) isolation of formaldehyde-crosslinked rat telomere nucleoprotein, (2) femtosecond UV laser crosslinking of telomere nucleoprotein inside of human nuclei, (3) mapping of the location and distribution of telomere proteins with respect to the ends of the telomere. We have shown that mammalian telomeres are organized as telomere-specific chromatin with closely spaced nucleosomes about 40 bp shorter than nucleosomes of bulk chromatin. In contrast to the situation in lower eukaryotes, nucleosomes compromise a major part of metazoan telomeres. However, nucleosomes are absent at the last kilobase of human telomeres. The protein TRF1 is present at the end of telomeres and interspersed with nucleosomes at the internal telomere regions. TRF2 is localized and binds to the last 600 bp of telomeres. We have proposed that human telomeres have telosome like structure at the last 1 kb.