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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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Chromosomal instability in wild-type, PARP-1+/−, and PARP-1−/− MEFs. (A) Cytogenetic alterations detected in PARP-1−/− metaphases from primary MEFs after hybridization with DAPI and a fluorescent Cy-3–labeled PNA-telomeric probe. For quantifications see Table I. Blue color corresponds to chromosome DNA stained with DAPI; yellow and white dots correspond to TTAGGG repeats. For definition of the different aberrations see Materials and methods. The different kinds of aberrations detected are indicated in the Figure. (B) Representative images of anaphase bridges in PARP-1−/− cells. Blue color corresponds to chromosome DNA stained with DAPI. (C) Representative image of a metaphase showing a typical Robertsonian translocation and the corresponding fragment with normal telomeres (indicated by arrows). (D) A metaphase spread showing a chromosome fusion in PARP-1−/− primary MEFs (indicated with an arrow). SKY detected that the fusion involves chromosomes X and 19. SKY analysis is shown as two insets: the top inset shows the same chromosome fusion after color classification, and the bottom inset shows the karyotype-arranged chromosomes with the direct fluorochrome image (left), DAPI counterstain (middle), and classified chromosomes (right). (E) A metaphase spread of PARP-1−/− MEFs showing the DAPI staining (left) and the SKY spectral image (right). The arrow indicates an association between chromosomes 6 and 10. A break was also present in the same rearrangement (the arrowhead points to a single break affecting chromosome 7).
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fig3: Chromosomal instability in wild-type, PARP-1+/−, and PARP-1−/− MEFs. (A) Cytogenetic alterations detected in PARP-1−/− metaphases from primary MEFs after hybridization with DAPI and a fluorescent Cy-3–labeled PNA-telomeric probe. For quantifications see Table I. Blue color corresponds to chromosome DNA stained with DAPI; yellow and white dots correspond to TTAGGG repeats. For definition of the different aberrations see Materials and methods. The different kinds of aberrations detected are indicated in the Figure. (B) Representative images of anaphase bridges in PARP-1−/− cells. Blue color corresponds to chromosome DNA stained with DAPI. (C) Representative image of a metaphase showing a typical Robertsonian translocation and the corresponding fragment with normal telomeres (indicated by arrows). (D) A metaphase spread showing a chromosome fusion in PARP-1−/− primary MEFs (indicated with an arrow). SKY detected that the fusion involves chromosomes X and 19. SKY analysis is shown as two insets: the top inset shows the same chromosome fusion after color classification, and the bottom inset shows the karyotype-arranged chromosomes with the direct fluorochrome image (left), DAPI counterstain (middle), and classified chromosomes (right). (E) A metaphase spread of PARP-1−/− MEFs showing the DAPI staining (left) and the SKY spectral image (right). The arrow indicates an association between chromosomes 6 and 10. A break was also present in the same rearrangement (the arrowhead points to a single break affecting chromosome 7).

Mentions: To study the impact of PARP-1 deficiency on telomere function, we analyzed the involvement of telomeres in the chromosomal aberrations spontaneously arising in true primary (passage 1) PARP-1−/− MEFs. For this, we performed Q-FISH of metaphasic nuclei with a fluorescent PNA-telomeric probe (Zijlmans et al., 1997) and then we scored for chromosomal aberrations (see “Q-FISH” in Materials and methods for description of different aberrations). Spontaneously arising chromosome aberrations were analyzed on at least 75 metaphases of each one of the primary (passage 1) wild-type, PARP-1+/−, and PARP-1−/− MEF cultures derived from heterozygous crosses (a total of 150, 225, and 375 metaphases for each genotype, respectively). As displayed in Table I, an increase in the frequency of chromosome/chromatid breaks and fragments was detected in primary MEFs isolated from PARP-1−/− or PARP-1+/− when compared with wild-type controls, 16.8, 16.8, and 8 events per 100 metaphases, respectively (Table I and Fig. 3 A). Primary MEFs isolated from PARP-1−/− or PARP-1+/− also showed a small increase in end-to-end fusions (Robertsonians+dicentrics+chromosome rings) compared with wild-type controls, 2.13, 2.22, and 0 fusions per 100 metaphases, respectively (Table I and Fig. 3 A). In some PARP-1−/− metaphases where Robertsonian-like fusions were detected, we were able to find the telomeric fragments resulting from the Robertsonian translocation (Fig. 3 C). These fragments always showed normal telomeres (Fig. 3 C), indicating that these fusions were the result of true Robertsonian translocations and not the consequence of telomere shortening. These Robertsonian fusions are different from those found in late generation telomerase-deficient mice that have critically short telomeres (Blasco et al., 1997). Furthermore, the fact that fusions detected in PARP-1−/− primary cells did not contain telomeres at the fusion point indicates that they are also different from those described in cells with impaired TRF2 function (van Steensel et al., 1998) or in cells deficient for nonhomologous end-joining DNA repair proteins such as Ku86 and DNA-PKc's (Bailey et al., 1999; Hsu et al., 2000; Samper et al., 2000; Goytisolo et al., 2001), all of which showed long telomeres at the fusion point (see Discussion).


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)

Chromosomal instability in wild-type, PARP-1+/−, and PARP-1−/− MEFs. (A) Cytogenetic alterations detected in PARP-1−/− metaphases from primary MEFs after hybridization with DAPI and a fluorescent Cy-3–labeled PNA-telomeric probe. For quantifications see Table I. Blue color corresponds to chromosome DNA stained with DAPI; yellow and white dots correspond to TTAGGG repeats. For definition of the different aberrations see Materials and methods. The different kinds of aberrations detected are indicated in the Figure. (B) Representative images of anaphase bridges in PARP-1−/− cells. Blue color corresponds to chromosome DNA stained with DAPI. (C) Representative image of a metaphase showing a typical Robertsonian translocation and the corresponding fragment with normal telomeres (indicated by arrows). (D) A metaphase spread showing a chromosome fusion in PARP-1−/− primary MEFs (indicated with an arrow). SKY detected that the fusion involves chromosomes X and 19. SKY analysis is shown as two insets: the top inset shows the same chromosome fusion after color classification, and the bottom inset shows the karyotype-arranged chromosomes with the direct fluorochrome image (left), DAPI counterstain (middle), and classified chromosomes (right). (E) A metaphase spread of PARP-1−/− MEFs showing the DAPI staining (left) and the SKY spectral image (right). The arrow indicates an association between chromosomes 6 and 10. A break was also present in the same rearrangement (the arrowhead points to a single break affecting chromosome 7).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196874&req=5

fig3: Chromosomal instability in wild-type, PARP-1+/−, and PARP-1−/− MEFs. (A) Cytogenetic alterations detected in PARP-1−/− metaphases from primary MEFs after hybridization with DAPI and a fluorescent Cy-3–labeled PNA-telomeric probe. For quantifications see Table I. Blue color corresponds to chromosome DNA stained with DAPI; yellow and white dots correspond to TTAGGG repeats. For definition of the different aberrations see Materials and methods. The different kinds of aberrations detected are indicated in the Figure. (B) Representative images of anaphase bridges in PARP-1−/− cells. Blue color corresponds to chromosome DNA stained with DAPI. (C) Representative image of a metaphase showing a typical Robertsonian translocation and the corresponding fragment with normal telomeres (indicated by arrows). (D) A metaphase spread showing a chromosome fusion in PARP-1−/− primary MEFs (indicated with an arrow). SKY detected that the fusion involves chromosomes X and 19. SKY analysis is shown as two insets: the top inset shows the same chromosome fusion after color classification, and the bottom inset shows the karyotype-arranged chromosomes with the direct fluorochrome image (left), DAPI counterstain (middle), and classified chromosomes (right). (E) A metaphase spread of PARP-1−/− MEFs showing the DAPI staining (left) and the SKY spectral image (right). The arrow indicates an association between chromosomes 6 and 10. A break was also present in the same rearrangement (the arrowhead points to a single break affecting chromosome 7).
Mentions: To study the impact of PARP-1 deficiency on telomere function, we analyzed the involvement of telomeres in the chromosomal aberrations spontaneously arising in true primary (passage 1) PARP-1−/− MEFs. For this, we performed Q-FISH of metaphasic nuclei with a fluorescent PNA-telomeric probe (Zijlmans et al., 1997) and then we scored for chromosomal aberrations (see “Q-FISH” in Materials and methods for description of different aberrations). Spontaneously arising chromosome aberrations were analyzed on at least 75 metaphases of each one of the primary (passage 1) wild-type, PARP-1+/−, and PARP-1−/− MEF cultures derived from heterozygous crosses (a total of 150, 225, and 375 metaphases for each genotype, respectively). As displayed in Table I, an increase in the frequency of chromosome/chromatid breaks and fragments was detected in primary MEFs isolated from PARP-1−/− or PARP-1+/− when compared with wild-type controls, 16.8, 16.8, and 8 events per 100 metaphases, respectively (Table I and Fig. 3 A). Primary MEFs isolated from PARP-1−/− or PARP-1+/− also showed a small increase in end-to-end fusions (Robertsonians+dicentrics+chromosome rings) compared with wild-type controls, 2.13, 2.22, and 0 fusions per 100 metaphases, respectively (Table I and Fig. 3 A). In some PARP-1−/− metaphases where Robertsonian-like fusions were detected, we were able to find the telomeric fragments resulting from the Robertsonian translocation (Fig. 3 C). These fragments always showed normal telomeres (Fig. 3 C), indicating that these fusions were the result of true Robertsonian translocations and not the consequence of telomere shortening. These Robertsonian fusions are different from those found in late generation telomerase-deficient mice that have critically short telomeres (Blasco et al., 1997). Furthermore, the fact that fusions detected in PARP-1−/− primary cells did not contain telomeres at the fusion point indicates that they are also different from those described in cells with impaired TRF2 function (van Steensel et al., 1998) or in cells deficient for nonhomologous end-joining DNA repair proteins such as Ku86 and DNA-PKc's (Bailey et al., 1999; Hsu et al., 2000; Samper et al., 2000; Goytisolo et al., 2001), all of which showed long telomeres at the fusion point (see Discussion).

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