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.

Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.

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...

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β 1 γ 2 subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.