XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions.

The major human AP endonuclease APE1 (HAP1, APEX, Ref1) initiates the repair of abasic sites generated either spontaneously, from attack of bases by free radicals, or during the course of the repair of damaged bases. APE1 therefore plays a central role in the base excision repair (BER) pathway. We report here that XRCC1, another essential protein involved in the maintenance of genome stability, physically interacts with APE1 and stimulates its enzymatic activities. A truncated form of APE1, lacking the first 35 amino acids, although catalytically proficient, loses the affinity for XRCC1 and is not stimulated by XRCC1. Chinese ovary cell lines mutated in XRCC1 have a diminished capacity to initiate the repair of AP sites. This defect is compensated by the expression of XRCC1. XRCC1, acting as both a scaffold and a modulator of the different activities involved in BER, would provide a physical link between the incision and sealing steps of the AP site repair process. The interaction described extends the coordinating role of XRCC1 to the initial step of the repair of DNA abasic sites.

Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step.

The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.