Biomedical subjects
Publications and source records attributed to Raymund J Wellinger.
Classically, the functions of linker histones (histones H1 and variants) have been related mainly to chromatin organization and the ensuing consequences on transcription. Remarkably, yeast histone H1 may not comply, as it appears to regulate homologous recombination specifically.
Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.
Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres. Furthermore, the lengths of telomeric repeats become very heterogeneous. These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends. To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity. The results show that double-mutant strains died more rapidly than strains lacking only telomerase components. Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27delta cells. The results from studies of the replication intermediates of a linear plasmid in rad27delta cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis. These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.
The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23 degrees C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30(ts). By several criteria, the Yku70-30p protein is functional at 23 degrees C and nonfunctional at 37 degrees C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase alpha gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.