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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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Telomere fluorescence distribution in wild-type, PARP-1+/−, and PARP-1−/− MEFs. Telomere length distribution in several different littermate wild-type (A10 and G4), PARP-1+/− (A7, E4, and G1), and PARP-1−/− (A6, E1, E2, G5, and G8) primary MEFs. The histogram depicts similar telomeres in all genotypes. One TFU corresponds to 1 Kb of TTAGGG repeats (see Table I and Table II for telomere length values in MEFs).
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fig1: Telomere fluorescence distribution in wild-type, PARP-1+/−, and PARP-1−/− MEFs. Telomere length distribution in several different littermate wild-type (A10 and G4), PARP-1+/− (A7, E4, and G1), and PARP-1−/− (A6, E1, E2, G5, and G8) primary MEFs. The histogram depicts similar telomeres in all genotypes. One TFU corresponds to 1 Kb of TTAGGG repeats (see Table I and Table II for telomere length values in MEFs).

Mentions: We studied whether PARP-1 deficiency affects the length of TTAGGG repeats at the telomeres using PARP-1−/− mice (Menissier-de Murcia et al., 1997). For this, littermate wild-type, PARP-1+/−, and PARP-1−/− mice or embryos derived from heterozygous crosses were used to quantify telomere length. Due to the fact that mouse telomeres show individual variability, it is essential to compare littermate mice, as well as to use several independent techniques to measure telomeres. Quantitative telomeric (Q)-FISH of wild-type, PARP-1+/− and PARP-1−/− littermate primary (passage 1) mouse embryonic fibroblasts (MEFs) revealed that all three genotypes showed a similar telomere length (Table I). The average telomere length was 35.24 ± 15.36, 40.29 ± 16.96, and 40.09 ± 15.85 Kb for wild-type (average of A10 and G4), PARP-1+/− (average of A7, E4, and G1), and PARP-1−/− (average of A6, E1, E2, G5, and G8) MEFs, respectively. These differences were not statistically significant (Student's t test, P > 0.01). It is noticeable that the standard deviations were also similar in the different genotypes, suggesting that telomeres were equally heterogeneous in length in all genotypes (see below). The Q-FISH data on MEFs was confirmed by using a different technique to measure telomere fluorescence based on Flow-FISH (described in Materials and methods; Table II). In this case, average telomere fluorescence expressed in arbitrary units was 3.51 ± 0.88, 3.69 ± 0.75, and 3.74 ± 0.67 for PARP-1+/+ (average of A10 and G4), PARP-1+/− (average of A7, E4, and G1), and PARP-1−/− (average of A6, E1, E2, G5, and G8) MEFs, respectively. Again, these differences were not statistically significant (Student's t tests, P > 0.01). Histograms showing the frequency of a given telomere fluorescence in littermate wild-type (average of A10 and G4; a total of 3,972 telomeres), PARP-1+/− (average of A7, E4, and G1; a total of 5,500 telomeres), and PARP-1−/− (average of A6, E1, E2, G5, and G8; a total of 8,772 telomeres) MEFs are presented in Fig. 1 . These histograms confirmed that the mean telomere fluorescence is similar in PARP-1−/−, PARP-1+/−, and wild-type MEFs, and furthermore showed that the heterogeneity of telomeric lengths is similar in both genotypes, ruling out significant differences in telomere length between 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)

Telomere fluorescence distribution in wild-type, PARP-1+/−, and PARP-1−/− MEFs. Telomere length distribution in several different littermate wild-type (A10 and G4), PARP-1+/− (A7, E4, and G1), and PARP-1−/− (A6, E1, E2, G5, and G8) primary MEFs. The histogram depicts similar telomeres in all genotypes. One TFU corresponds to 1 Kb of TTAGGG repeats (see Table I and Table II for telomere length values in MEFs).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196874&req=5

fig1: Telomere fluorescence distribution in wild-type, PARP-1+/−, and PARP-1−/− MEFs. Telomere length distribution in several different littermate wild-type (A10 and G4), PARP-1+/− (A7, E4, and G1), and PARP-1−/− (A6, E1, E2, G5, and G8) primary MEFs. The histogram depicts similar telomeres in all genotypes. One TFU corresponds to 1 Kb of TTAGGG repeats (see Table I and Table II for telomere length values in MEFs).
Mentions: We studied whether PARP-1 deficiency affects the length of TTAGGG repeats at the telomeres using PARP-1−/− mice (Menissier-de Murcia et al., 1997). For this, littermate wild-type, PARP-1+/−, and PARP-1−/− mice or embryos derived from heterozygous crosses were used to quantify telomere length. Due to the fact that mouse telomeres show individual variability, it is essential to compare littermate mice, as well as to use several independent techniques to measure telomeres. Quantitative telomeric (Q)-FISH of wild-type, PARP-1+/− and PARP-1−/− littermate primary (passage 1) mouse embryonic fibroblasts (MEFs) revealed that all three genotypes showed a similar telomere length (Table I). The average telomere length was 35.24 ± 15.36, 40.29 ± 16.96, and 40.09 ± 15.85 Kb for wild-type (average of A10 and G4), PARP-1+/− (average of A7, E4, and G1), and PARP-1−/− (average of A6, E1, E2, G5, and G8) MEFs, respectively. These differences were not statistically significant (Student's t test, P > 0.01). It is noticeable that the standard deviations were also similar in the different genotypes, suggesting that telomeres were equally heterogeneous in length in all genotypes (see below). The Q-FISH data on MEFs was confirmed by using a different technique to measure telomere fluorescence based on Flow-FISH (described in Materials and methods; Table II). In this case, average telomere fluorescence expressed in arbitrary units was 3.51 ± 0.88, 3.69 ± 0.75, and 3.74 ± 0.67 for PARP-1+/+ (average of A10 and G4), PARP-1+/− (average of A7, E4, and G1), and PARP-1−/− (average of A6, E1, E2, G5, and G8) MEFs, respectively. Again, these differences were not statistically significant (Student's t tests, P > 0.01). Histograms showing the frequency of a given telomere fluorescence in littermate wild-type (average of A10 and G4; a total of 3,972 telomeres), PARP-1+/− (average of A7, E4, and G1; a total of 5,500 telomeres), and PARP-1−/− (average of A6, E1, E2, G5, and G8; a total of 8,772 telomeres) MEFs are presented in Fig. 1 . These histograms confirmed that the mean telomere fluorescence is similar in PARP-1−/−, PARP-1+/−, and wild-type MEFs, and furthermore showed that the heterogeneity of telomeric lengths is similar in both genotypes, ruling out significant differences in telomere length between 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