Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 ± 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3 overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.

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because of difficulties in reproducing our initial findings, we investigated the possibility that there might have been contamination by plasmid DNA derived from E. coli in the DNA preparations extracted from N. crassa. To determine whether this was the case, the DNAs from the original isolates were incubated with restriction endonuclease Bcl I and examined for specific restriction enzyme cleavage products. Southern blot analysis revealed that a substantial fraction of the plasmid present in these isolates was resistant to Bcl I digestion whereas the N. crassa genomic DNAs and inserted plasmid sequences were completely digested by RM I. Because Bcl I does not digest DNA that has been methylated by the E. coli dam methylase, we interpret this as evidence for plasmid contamination because there is no comparable DNA modification enzyme in N. crassa. Furthermore, in subsequent experiments using DNAs extracted from N. crassa transformants that show no evidence of contamination, we have failed to recover pDV1001 by transformation of E. coli. Although the fact that pDV1001 produces "abortive transformants" suggests that it carries an ars sequence, we now have no evidence to suggest that it is capable of sufficient stable autonomous replication without integration into the N. crassa genome to prove useful as a potential "shuttle" vector between N. crassa and E. coli.Correction. The article "Bent helical structure in kinetoplast DNA"

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Most normal diploid human cells do not express telomerase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the length of the single-stranded G-rich telomeric 3-overhang is proportional to the rate of shortening in four human cell types that exhibit different rates of telomere shortening in culture. These results provide direct evidence that the size of the G-rich overhang is not fixed but subject to regulation. The potential ability to manipulate this rate has profound implications both for slowing the rate of replicative aging in normal cells and for accelerating the rate of telomere loss in cancer cells in combination with anti-telomerase therapies.Telomerase is not expressed in most normal tissues but is present in 85-90% of all human tumors (1), and there is considerable interest in the potential oncologic use of telomerase inhibitors. One concern is that such inhibitors would not directly kill tumor cells but only initiate telomere shortening, and thus it might take many cell divisions before a therapeutic effect occurred. Cultured human cells exhibit different rates of telomere shortening (2-5), implying that this rate is not fixed but might be subject to manipulation. Agents that accelerate the rate of shortening might greatly augment the efficacy of anti-telomerase treatments. However, virtually nothing is known about what controls the rate of telomere shortening in normal telomerase-negative human cells.Telomeres of eukaryotic cells contain G-rich single-stranded 3Ј-overhangs, which extend beyond the double-stranded region. While the exact structure of these overhangs varies between species, the presence of overhangs is both conserved and believed to be essential for the maintenance of chromosome end structure and function. Studies in ciliates and yeast indicate that end-processing activities include 5Ј-nucleases that digest the C-rich telomeric strand, telomerase that elongates the Grich strand, nucleases that trim the G-rich strand so that it ends at a nucleotide other than the normal telomerase pause site, and activities that fill-in the C-rich strand (6 -11). DNA polymerases ␣ and ␦ and primase are all required for telomerase activity in Saccharomyces cerevisiae (12), supporting the concept that this fill-in activity is carried out by the conventional lagging strand synthetic machinery (13). The 12-14-nucleotide single-stranded G-rich 3Ј-overhang in hypotrichous ciliate telomeres (14) and the identification of a primase activity that can initiate DNA synthesis at the very 3Ј-end of the G-rich strand (15, 16) have led to the concept that the overhang is produced following digestion of a terminally positioned RNA primer. Telomeres of yeast mutants lacking telomerase shorten by only 3-5 bp 1 per division (17), showing that even in the absence of telomerase yeast end-processing activities are able to replicate all but a few nucleotides at the end of the telomere. In contrast, rates of telomere shortening in human cells lacking telomerase can vary from 30 to s...