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Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway
R A Bennett1, D M Wilson, D Wong
1Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115, USA.
Abstract:
Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase beta (Polbeta) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Polbeta and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polbeta into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polbeta exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5'-terminal deoxyribose 5-phosphate by Polbeta. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.
Insights
DNA repair involves coordinated action between AP endonuclease and DNA polymerase beta. These proteins interact to efficiently correct abasic sites, ensuring DNA integrity and genomic stability.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Biochemistry
Background:
- Abasic (AP) sites are mutagenic lesions crucial in DNA damage and repair.
- Base excision repair (BER) pathway corrects AP sites through incision, excision, synthesis, and ligation.
- Mammalian DNA polymerase beta (Polbeta) and AP endonuclease (Ape) are key enzymes in BER.
Purpose of the Study:
- To investigate the protein-protein interactions between Polbeta and Ape.
- To elucidate the functional coordination of Polbeta and Ape in AP site repair.
Main Methods:
- Yeast two-hybrid analysis to detect protein-protein interactions.
- In vitro binding assays to study complex formation.
- Kinetic experiments to measure enzyme activity and reaction rates.
Main Results:
- Yeast two-hybrid analysis revealed direct contact between Polbeta and Ape.
- Ape protein binding to AP sites facilitates Polbeta loading into a ternary complex.
- Polbeta exhibits stable DNA binding post-incision, and Ape accelerates Polbeta's deoxyribose excision.
Conclusions:
- Polbeta and Ape physically interact and coordinate their functions in sequential steps of BER.
- This coordination ensures efficient correction of abasic sites, maintaining genomic stability.