Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

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DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repair in vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase ␤-dependent single nucleotide insertion pathway. DNA base excision repair (BER)1 counteracts the mutagenic and cytotoxic effects of various kinds of base alterations that do not significantly distort the secondary structure of the double helix. A common intermediate of this pathway is the abasic (AP) site, that arises as a consequence of removal of altered bases by DNA-N-glycosylases or as spontaneous detachment of normal bases from the deoxyribose-phosphate backbone. It has been calculated that 2000 -10000 AP sites arise each day in a mammalian cell under physiological conditions (1). Therefore, the task of BER is engaging and important, and data obtained in Escherichia coli and transgenic mice show that this process is essential for survival (2-4). We have recently shown that, in addition to the polymerase ␤-dependent single nucleotide insertion pathway previously investigated in mammalian cells (5), a distinct proliferating cell nuclear antigen (PCNA)-dependent pathway is also present that incorporates a repair patch size of 7-14 nucleotides extending 3Ј to the site of the lesion (6). Our knowledge of the enzymology of the two pathways has several gaps. In particular, the enzymology of the ligation step is poorly defined. A role for the XRCC1 protein has been suggested on the basis of the sensitivity of xrcc1 mutant cell lines (the CHO derivatives EM9 and EM-C11) to agents that introduce DNA base damage (7, 8) and because of their reduced rate of single-strand break rejoining following exposure to ionizing radiation or alkylating agents (9, 10). Consistent with a role for XRCC1 in DNA ligation and BER is its observed interaction with DNA ligase III and DNA polymerase ␤ (7, 11, 12). Here, we have examined directly the role of XRCC1 and DNA ligase III in mammalian BER using a cellfree system. We report for the first time that (i) xrcc1 mutant cells are partially defective in ligation of BER patches and (ii) the defect involves only the polymerase ␤-dependent single nucle...

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