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Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase-deficient mice and primary cells despite increased chromosomal instability.

Samper E, Goytisolo FA, Ménissier-de Murcia J, González-Suárez E, Cigudosa JC, de Murcia G, Blasco MA - J. Cell Biol. (2001)

Bottom Line: Similarly, there were no differences in the length of the G-strand overhang.The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency.Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus Cantoblanco, E-28049 Madrid, Spain.

ABSTRACT
Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1-deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1-deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. NAT: Genet. 23:76-80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1(-/)- littermate mice or PARP-1(-/)- primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1(-/)- cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1(-/)- primary cells is accordant with the absence of severe proliferative defects in PARP-1(-/)- mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

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Normal G-strand overhang in PARP-1−/− primary cells. G-strand overhangs in littermate wild-type and PARP-1−/− MEFs are visualized in native gel after hybridization with a (CCCTAA)4 probe (see Materials and methods). Notice that upon treatment with two different doses of mung bean nuclease (40 and 100 U) the G-strand–specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. A10 and A6 are littermate wild-type and PARP-1−/− MEFs, respectively. G4 and G8 are littermate primary MEF cultures.
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fig4: Normal G-strand overhang in PARP-1−/− primary cells. G-strand overhangs in littermate wild-type and PARP-1−/− MEFs are visualized in native gel after hybridization with a (CCCTAA)4 probe (see Materials and methods). Notice that upon treatment with two different doses of mung bean nuclease (40 and 100 U) the G-strand–specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. A10 and A6 are littermate wild-type and PARP-1−/− MEFs, respectively. G4 and G8 are littermate primary MEF cultures.

Mentions: G-strand overhangs are regions of G-rich single-stranded telomeric DNA that protrude in the 3′ direction from the double-stranded telomere. These single-stranded G-rich regions have been involved recently in the formation of a special structure at the chromosome end named T-loop, which has been proposed to protect the ends from recombination and DNA repair activities (Griffith et al., 1999). Hence, examination of telomeric G-strand overhangs in PARP-1 cells is crucial to gain more insight into the telomere integrity of these cells. To study the telomeric G-strand overhangs, we carried out TRF analysis with a (CCCTAA)4 probe as described (Samper et al., 2000) using nondenaturing pulse field agarose gels (Materials and methods). Detection of a signal with the (CCCTAA)4 probe hybridized to native DNA samples indicates the presence of the G-strand overhang. Primary wild-type and PARP-1−/− MEFs showed G-strand–specific signals that were similar in size and intensity in all genotypes (Fig. 4) . Table V shows quantification of the G-strand signals; the wild-type values were normalized to 100 in each litter. The average G-strand signal for PARP-1−/− MEFs was 120% that of the wild-types; however, this difference was not statistically significant (Student's t test, P = 0.24). To show that the probe specifically recognized the single-stranded telomeric tail, treatment with mung bean nuclease that specifically degrades single-stranded DNA and RNA overhangs was performed. As expected, the G-strand signal decreased in all genotypes upon treatment, as shown in Fig. 4 (“native gel”). As control, the same gel was denatured and rehybridized with the (CCCTAA)4 probe, which highlighted the TRFs (Fig. 4; “denaturing gel”), again showing no difference in TRF lengths between wild-type and PARP-1−/− genotypes.


Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase-deficient mice and primary cells despite increased chromosomal instability.

Samper E, Goytisolo FA, Ménissier-de Murcia J, González-Suárez E, Cigudosa JC, de Murcia G, Blasco MA - J. Cell Biol. (2001)

Normal G-strand overhang in PARP-1−/− primary cells. G-strand overhangs in littermate wild-type and PARP-1−/− MEFs are visualized in native gel after hybridization with a (CCCTAA)4 probe (see Materials and methods). Notice that upon treatment with two different doses of mung bean nuclease (40 and 100 U) the G-strand–specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. A10 and A6 are littermate wild-type and PARP-1−/− MEFs, respectively. G4 and G8 are littermate primary MEF cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196874&req=5

fig4: Normal G-strand overhang in PARP-1−/− primary cells. G-strand overhangs in littermate wild-type and PARP-1−/− MEFs are visualized in native gel after hybridization with a (CCCTAA)4 probe (see Materials and methods). Notice that upon treatment with two different doses of mung bean nuclease (40 and 100 U) the G-strand–specific signal decreases. As control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. A10 and A6 are littermate wild-type and PARP-1−/− MEFs, respectively. G4 and G8 are littermate primary MEF cultures.
Mentions: G-strand overhangs are regions of G-rich single-stranded telomeric DNA that protrude in the 3′ direction from the double-stranded telomere. These single-stranded G-rich regions have been involved recently in the formation of a special structure at the chromosome end named T-loop, which has been proposed to protect the ends from recombination and DNA repair activities (Griffith et al., 1999). Hence, examination of telomeric G-strand overhangs in PARP-1 cells is crucial to gain more insight into the telomere integrity of these cells. To study the telomeric G-strand overhangs, we carried out TRF analysis with a (CCCTAA)4 probe as described (Samper et al., 2000) using nondenaturing pulse field agarose gels (Materials and methods). Detection of a signal with the (CCCTAA)4 probe hybridized to native DNA samples indicates the presence of the G-strand overhang. Primary wild-type and PARP-1−/− MEFs showed G-strand–specific signals that were similar in size and intensity in all genotypes (Fig. 4) . Table V shows quantification of the G-strand signals; the wild-type values were normalized to 100 in each litter. The average G-strand signal for PARP-1−/− MEFs was 120% that of the wild-types; however, this difference was not statistically significant (Student's t test, P = 0.24). To show that the probe specifically recognized the single-stranded telomeric tail, treatment with mung bean nuclease that specifically degrades single-stranded DNA and RNA overhangs was performed. As expected, the G-strand signal decreased in all genotypes upon treatment, as shown in Fig. 4 (“native gel”). As control, the same gel was denatured and rehybridized with the (CCCTAA)4 probe, which highlighted the TRFs (Fig. 4; “denaturing gel”), again showing no difference in TRF lengths between wild-type and PARP-1−/− genotypes.

Bottom Line: Similarly, there were no differences in the length of the G-strand overhang.The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency.Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus Cantoblanco, E-28049 Madrid, Spain.

ABSTRACT
Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1-deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1-deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. NAT: Genet. 23:76-80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1(-/)- littermate mice or PARP-1(-/)- primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1(-/)- cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1(-/)- primary cells is accordant with the absence of severe proliferative defects in PARP-1(-/)- mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

Show MeSH
Related in: MedlinePlus