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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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Telomerase activity in wild-type, PARP-1+/−, and PARP-1−/− MEFs. S-100 extracts were prepared from wild-type (A10 and G1), PARP-1+/− (A7), and PARP−/− (A6, G8, and E1) primary MEFs cultures and assayed for telomerase activity. Extracts were pretreated (+) or not (−) with RNase. Protein concentration used is indicated. The arrow indicates the internal control (IC) for PCR efficiency. Same letter refers to littermate embryos.
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fig5: Telomerase activity in wild-type, PARP-1+/−, and PARP-1−/− MEFs. S-100 extracts were prepared from wild-type (A10 and G1), PARP-1+/− (A7), and PARP−/− (A6, G8, and E1) primary MEFs cultures and assayed for telomerase activity. Extracts were pretreated (+) or not (−) with RNase. Protein concentration used is indicated. The arrow indicates the internal control (IC) for PCR efficiency. Same letter refers to littermate embryos.

Mentions: A direct interaction between PARP-1 and DNA polymerase α-primase tetramer has been demonstrated through the catalytic subunit of DNA polymerase α and the PARP-1 DNA binding domain, suggesting a link between DNA strand break detection and DNA replication (Simbulan-Rosenthal et al., 1996; Dantzer et al., 1998). Since telomerase is the cellular reverse transcriptase involved in replicating telomeric DNA (for review see Nugent and Lundblad, 1998), we investigated whether the catalytic subunit of telomerase, Tert, could also interact or be a target of PARP-1 activity. For this, we first determined telomerase activity levels in wild-type and in PARP-1−/− cells which lack PARP-1 activity. As shown in Fig. 5 , telomerase activity levels are similar in wild-type (A10 and G1), PARP-1+/− (A7), and PARP−/− (A6, G8, and E1) primary MEF cultures, indicating that lack of PARP-1 activity does not significantly affect the levels of telomerase activity in the cell. A similar absence of changes in telomerase activity between wild-type and PARP-1−/− cells was obtained upon treatment of cells with genotoxic agents such as gamma irradiation and H2O2 which dramatically activate PARP-1 (not shown).


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)

Telomerase activity in wild-type, PARP-1+/−, and PARP-1−/− MEFs. S-100 extracts were prepared from wild-type (A10 and G1), PARP-1+/− (A7), and PARP−/− (A6, G8, and E1) primary MEFs cultures and assayed for telomerase activity. Extracts were pretreated (+) or not (−) with RNase. Protein concentration used is indicated. The arrow indicates the internal control (IC) for PCR efficiency. Same letter refers to littermate embryos.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Telomerase activity in wild-type, PARP-1+/−, and PARP-1−/− MEFs. S-100 extracts were prepared from wild-type (A10 and G1), PARP-1+/− (A7), and PARP−/− (A6, G8, and E1) primary MEFs cultures and assayed for telomerase activity. Extracts were pretreated (+) or not (−) with RNase. Protein concentration used is indicated. The arrow indicates the internal control (IC) for PCR efficiency. Same letter refers to littermate embryos.
Mentions: A direct interaction between PARP-1 and DNA polymerase α-primase tetramer has been demonstrated through the catalytic subunit of DNA polymerase α and the PARP-1 DNA binding domain, suggesting a link between DNA strand break detection and DNA replication (Simbulan-Rosenthal et al., 1996; Dantzer et al., 1998). Since telomerase is the cellular reverse transcriptase involved in replicating telomeric DNA (for review see Nugent and Lundblad, 1998), we investigated whether the catalytic subunit of telomerase, Tert, could also interact or be a target of PARP-1 activity. For this, we first determined telomerase activity levels in wild-type and in PARP-1−/− cells which lack PARP-1 activity. As shown in Fig. 5 , telomerase activity levels are similar in wild-type (A10 and G1), PARP-1+/− (A7), and PARP−/− (A6, G8, and E1) primary MEF cultures, indicating that lack of PARP-1 activity does not significantly affect the levels of telomerase activity in the cell. A similar absence of changes in telomerase activity between wild-type and PARP-1−/− cells was obtained upon treatment of cells with genotoxic agents such as gamma irradiation and H2O2 which dramatically activate PARP-1 (not shown).

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