The repair pathways involved in the removal of thymine glycol (TG) from DNA by human cell extracts have been examined. Closed circular DNA constructs containing a single TG at a defined site were used as substrates to determine the patch size generated after in vitro repair by cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that the majority of TG was repaired through the base excision repair (BER) pathways. Repair incorporation 5 to the lesion, characteristic for the nucleotide excision repair pathway, was not found. More than 80% of the TG repair was accomplished by the single-nucleotide repair mechanism, and the remaining TGs were removed by the long patch BER pathway. We also analyzed the role of the xeroderma pigmentosum, complementation group G (XPG) protein in the excision step of BER. Cell extracts deficient in XPG protein had an average 25% reduction in TG incision. These data show that BER is the primary pathway for repair of TG in DNA and that XPG protein may be involved in repair of TG as an accessory factor.

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G°) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.

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