Role of mammalian Rad54 in telomere length maintenance - PubMed (original) (raw)

Role of mammalian Rad54 in telomere length maintenance

Isabel Jaco et al. Mol Cell Biol. 2003 Aug.

Free PMC article

Abstract

The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.

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Figures

FIG. 1.

FIG. 1.

Telomere length analysis by Q-FISH. (A) Average telomere fluorescence and standard error of q-telomeres, p-telomeres, or all (p+q) telomeres in primary MEFs grouped by genotype. Fluorescence is expressed in telomere fluorescence units (TFUs), where 1 TFU corresponds to 1 kb of TTAGGG repeats (30). Each value represents the mean of 10 metaphases and of the indicated number (n) of individual telomere values. n refers to the total number of telomere values used for calculation of the average telomere fluorescence for each chromosome arm, as well as for the sum of both arms. Bars: ▪, average of q- and p-telomeres; formula image, q-telomeres; □, p-telomeres. Despite the wide heterogeneity in individual telomere fluorescence intensity values (see, for example, Fig. 1B), the standard errors of the mean are very small due to the large number of datum points (see “_n_” values). As a result, the error bars are not visible in the graphs (see Table 1 for standard error values). (B) Histograms showing the telomere length frequencies for p-arms, q-arms, or the sum of p+q-arms of primary MEFs grouped by genotype. The letters indicate the individual MEFs of each genotype used for the analysis. n is the total number of p-telomeres, q-telomeres, or the sum of p+q-telomeres per genotype that are represented in each histogram. One TFU corresponds to 1 kb of TTAGGG repeats (30). To facilitate visualization of the telomere length values, two vertical lines indicate the position of the 20- and 60-kb telomeres for each histogram. The telomere length frequency distribution in each histogram is an indication of the standard deviation of telomere length values and not of the standard error. (C) Percentage of telomeres of the total number of telomeres analyzed for each genotype that are ≤20 kb or ≥60 kb, as indicated. The absolute numbers of telomeres used for the analysis are also shown on top of the bars.

FIG. 2.

FIG. 2.

Telomerase activity in MEFs. S-100 extracts were prepared from MEFs of the indicated genotype and assayed for telomerase activity. For some genotypes more than 1 MEF was assayed for TRAP activity. Different letters refer to independent MEFs. Extracts were pretreated (+R) or not with RNase. The protein concentration used is indicated. The arrow indicates the internal control (IC) for PCR efficiency.

FIG. 3.

FIG. 3.

CO-FISH primary MEFs. (Left panel) Frequency of chromosome-type or chromatid-type fusions involving telomeres produced by leading-strand synthesis in metaphases of the indicated genotypes. (+)TTAGGG, fusions showing TTAGGG signal at the fusion point; (−)TTAGGG, fusions lacking TTAGGG signal at the fusion point. (Right panels) Representative CO-FISH images of chromosome-type telomere fusions containing TTAGGG repeats at the fusion point (yellow arrows) in the indicated individual MEF. Blue, DAPI; red, TTAGGG signal.

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