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Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

Croset A, Cordelières FP, Berthault N, Buhler C, Sun JS, Quanz M, Dutreix M - Nucleic Acids Res. (2013)

Bottom Line: We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation.Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP.Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, CNRS-UMR3347, INSERM-U1021, 91405 Orsay, France, DNA Therapeutics, Génopole, 91000 Evry, France, Institut Curie, CNRS-UMR3348, Plateforme PICT-IBiSA, 91405 Orsay, France and Museum National d'Histoire Naturelle, USM503, 75231 Paris, France.

ABSTRACT
One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

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PARP and DNA-PK activation induced by siDNA. PARylation and DNA-PK kinase activities were measured either with purified enzymes (A, B and E) or nuclear extracts from MRC5 cells (C, D and F): kinase activity was estimated by measuring P53 peptide phosphorylation after addition of 0.5 µM siDNA (A, C). PARP activation was estimated by measuring H1 histone parylation after addition of 0.2 µM siDNA (B, D) or increasing amounts of siDNA (Panel E, F: Pbait32, triangle; Pbait12, square; Dbait8H, diamond). Reported values represent the mean value and standard deviation of at least three independent experiments. Two 32-bp-long Pbait molecules with different sequences were tested (Pbait32 and Pbait32L). The Dbait8H and Bait32C were used as negative control.
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gkt522-F2: PARP and DNA-PK activation induced by siDNA. PARylation and DNA-PK kinase activities were measured either with purified enzymes (A, B and E) or nuclear extracts from MRC5 cells (C, D and F): kinase activity was estimated by measuring P53 peptide phosphorylation after addition of 0.5 µM siDNA (A, C). PARP activation was estimated by measuring H1 histone parylation after addition of 0.2 µM siDNA (B, D) or increasing amounts of siDNA (Panel E, F: Pbait32, triangle; Pbait12, square; Dbait8H, diamond). Reported values represent the mean value and standard deviation of at least three independent experiments. Two 32-bp-long Pbait molecules with different sequences were tested (Pbait32 and Pbait32L). The Dbait8H and Bait32C were used as negative control.

Mentions: siDNA molecules were screened for their ability to recruit PARP and induce the synthesis of a poly (ADP-ribose) chain (PAR) referred to as PARylation (Figure 2). PARP activity assays were performed using purified PARP enzyme (Figure 2B and E) and MRC5 cell extracts (Figure 2D and F). Dbait32Hc and Pbait32 molecules (32 bp) efficiently activated both purified PARP and PARP in crude extracts. These results confirm previous observations that PARP binds to DSB with a high affinity (22). Shorter Dbait or Pbait molecules did not efficiently activate PARP in crude extract. Analysis of PARP activation as a function of siDNA concentration showed that maximal activation required 10-fold more Pbait12 than Pbait32 (Figure 2E and F). As expected, Bait32C molecules that have no nick or free ends did not activate PARP. Pbait32L and Pbait32 that have the same structure but differ in DNA sequence (Figure 1) had similar PARP activation activities. PARP activation thus did not depend on the DNA sequence. Moreover, only Dbait and not Pbait activated DNA-PK whether as a purified enzyme (Figure 2A) or in MRC5 cell extracts (Figure 2C). This result is in agreement with previous observation that 34–32-bp long dumbbell form with no free ends does not bind DNA-PK (27). We chose the 32-bp-long Pbait and Dbait molecules (hereafter called Pbait32 and Dbait32Hc), which activated PARP and PARP/DNA-PK, respectively, for further studies in cell cultures. We used an 8-bp-long Dbait (Dbait8H), which does not activate DNA-PK or PARP, as a transfection control (Figure 2).Figure 2.


Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

Croset A, Cordelières FP, Berthault N, Buhler C, Sun JS, Quanz M, Dutreix M - Nucleic Acids Res. (2013)

PARP and DNA-PK activation induced by siDNA. PARylation and DNA-PK kinase activities were measured either with purified enzymes (A, B and E) or nuclear extracts from MRC5 cells (C, D and F): kinase activity was estimated by measuring P53 peptide phosphorylation after addition of 0.5 µM siDNA (A, C). PARP activation was estimated by measuring H1 histone parylation after addition of 0.2 µM siDNA (B, D) or increasing amounts of siDNA (Panel E, F: Pbait32, triangle; Pbait12, square; Dbait8H, diamond). Reported values represent the mean value and standard deviation of at least three independent experiments. Two 32-bp-long Pbait molecules with different sequences were tested (Pbait32 and Pbait32L). The Dbait8H and Bait32C were used as negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753643&req=5

gkt522-F2: PARP and DNA-PK activation induced by siDNA. PARylation and DNA-PK kinase activities were measured either with purified enzymes (A, B and E) or nuclear extracts from MRC5 cells (C, D and F): kinase activity was estimated by measuring P53 peptide phosphorylation after addition of 0.5 µM siDNA (A, C). PARP activation was estimated by measuring H1 histone parylation after addition of 0.2 µM siDNA (B, D) or increasing amounts of siDNA (Panel E, F: Pbait32, triangle; Pbait12, square; Dbait8H, diamond). Reported values represent the mean value and standard deviation of at least three independent experiments. Two 32-bp-long Pbait molecules with different sequences were tested (Pbait32 and Pbait32L). The Dbait8H and Bait32C were used as negative control.
Mentions: siDNA molecules were screened for their ability to recruit PARP and induce the synthesis of a poly (ADP-ribose) chain (PAR) referred to as PARylation (Figure 2). PARP activity assays were performed using purified PARP enzyme (Figure 2B and E) and MRC5 cell extracts (Figure 2D and F). Dbait32Hc and Pbait32 molecules (32 bp) efficiently activated both purified PARP and PARP in crude extracts. These results confirm previous observations that PARP binds to DSB with a high affinity (22). Shorter Dbait or Pbait molecules did not efficiently activate PARP in crude extract. Analysis of PARP activation as a function of siDNA concentration showed that maximal activation required 10-fold more Pbait12 than Pbait32 (Figure 2E and F). As expected, Bait32C molecules that have no nick or free ends did not activate PARP. Pbait32L and Pbait32 that have the same structure but differ in DNA sequence (Figure 1) had similar PARP activation activities. PARP activation thus did not depend on the DNA sequence. Moreover, only Dbait and not Pbait activated DNA-PK whether as a purified enzyme (Figure 2A) or in MRC5 cell extracts (Figure 2C). This result is in agreement with previous observation that 34–32-bp long dumbbell form with no free ends does not bind DNA-PK (27). We chose the 32-bp-long Pbait and Dbait molecules (hereafter called Pbait32 and Dbait32Hc), which activated PARP and PARP/DNA-PK, respectively, for further studies in cell cultures. We used an 8-bp-long Dbait (Dbait8H), which does not activate DNA-PK or PARP, as a transfection control (Figure 2).Figure 2.

Bottom Line: We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation.Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP.Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, CNRS-UMR3347, INSERM-U1021, 91405 Orsay, France, DNA Therapeutics, Génopole, 91000 Evry, France, Institut Curie, CNRS-UMR3348, Plateforme PICT-IBiSA, 91405 Orsay, France and Museum National d'Histoire Naturelle, USM503, 75231 Paris, France.

ABSTRACT
One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

Show MeSH
Related in: MedlinePlus