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Inhibition of poly(ADP-ribose) synthesis may affect DNA repair prior to ligation
Abstract:
The effects of modification of poly(ADP-ribosyl)ation reactions have been examined in normal (F107) and ataxia telangiectasia (AT23) fibroblasts following damage by methyl methanesulphonate (MMS) and u.v. light. The technique of benzoylated DEAE (BD)-cellulose chromatography was utilized to estimate both the extent and nature of the damage to DNA induced by these agents and to examine the effects of an inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), on these parameters. Single strand breakage, determined by nucleoid sedimentation, and levels of poly ADP(ribose) synthesis were monitored. Increase in the proportion of DNA containing single-stranded regions, as measured by stepwise elution from BD-cellulose, was observed following MMS damage in both cell types. In the presence of 3AB, a further accumulation of DNA containing single-stranded regions occurred, with the effect being more prominent in AT23 fibroblasts. U.v. light damage did not induce increased binding to BD-cellulose in normal cells, and the increase observed in AT23 cells was much less than that seen following alkylation damage. Examination of the nature of single-stranded damage by caffeine gradient elution from BD-cellulose following MMS treatment revealed discrete structural lesions, which were enhanced in the presence of 3AB. A similar effect was exerted by arabinofuranosyl cytosine. The behaviour of these intermediates, which could be associated with repair, was not in accord with the suggestion that 3AB inhibits only the ligation stage of the repair process. Our results suggest that specific intermediate stages in DNA repair are sensitive to 3AB, and it seems likely that these stages occur prior to ligation.
Insights
The study investigated how inhibiting poly(ADP-ribose) synthetase with 3-aminobenzamide affects DNA repair in normal and ataxia telangiectasia cells after methyl methanesulphonate damage. Results show 3-aminobenzamide targets early DNA repair stages before ligation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Poly(ADP-ribosyl)ation is a crucial post-translational modification involved in DNA repair.
- Ataxia telangiectasia (AT) is a genetic disorder characterized by DNA repair deficiencies.
- Methyl methanesulphonate (MMS) and u.v. light are DNA-damaging agents used to study repair mechanisms.
Purpose of the Study:
- To investigate the effects of inhibiting poly(ADP-ribose) synthetase on DNA repair pathways.
- To compare DNA repair responses in normal and AT fibroblasts after exposure to MMS and u.v. light.
- To elucidate the role of 3-aminobenzamide (3AB) in DNA damage processing.
Main Methods:
- Benzoylated DEAE (BD)-cellulose chromatography to assess DNA damage and repair.
- Nucleoid sedimentation to determine single-strand breaks.
- Monitoring poly ADP(ribose) synthesis levels.
- Treatment with 3-aminobenzamide (3AB) and arabinofuranosyl cytosine.
Main Results:
- MMS induced DNA single-strand breaks and single-stranded regions in both normal and AT cells.
- 3AB exacerbated the accumulation of single-stranded DNA regions, particularly in AT cells.
- U.v. light caused less DNA damage compared to MMS, with minimal effect in normal cells.
- Discrete DNA lesions sensitive to 3AB were identified, suggesting involvement in early repair stages.
Conclusions:
- 3-aminobenzamide inhibits specific intermediate stages of DNA repair, occurring before ligation.
- The DNA repair process in AT fibroblasts is more sensitive to 3AB-induced inhibition.
- The findings challenge the notion that 3AB solely inhibits the ligation step in DNA repair.