Two Pathways for Base Excision Repair in Mammalian Cells

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. 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.