Two Pathways for Base Excision Repair in Mammalian Cells

Abstract

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.

Mammalian base excision repair by DNA polymerases δ and ε

“…We have previously shown that the short-patch BER in wild-type cell extracts is PCNA-independent (Frosina et al, 1996) and in Pol b-de®cient cells is slightly stimulated by PCNA (Fortini et al, 1998). The repair e ciency of Pol d-and Pol e-containing fractions was investigated in the presence of increasing amounts of PCNA (Figure 4).…”

“…The incorporation of radiolabeled dTMP in the BamHI ± HindIII restriction fragment marks mainly the occurrence of one-gap ®lling reactions that are the major route for repair of AP sites, while the incorporation of dCMP identi®es exclusively 2-6 nucleotides repair patches. Moreover since PCNA is required for the long-patch but not for the short-patch BER (Frosina et al, 1996;Fortini et al, 1998) the dependence of the repair reaction on this auxiliary protein is an additional marker to discriminate between the two pathways.…”

“…A typical feature of the long-patch BER is that it requires PCNA (Matsumoto et al, 1994;Frosina et al, 1996;Klungland and Lindahl, 1997). The ability of Pol d-and Pol e-containing fractions to perform long-patch repair synthesis was analysed in the presence of increasing concentrations of PCNA ( Figure 5).…”

“…In contrast, however, the identity of the Pols involved in the long-patch BER is still matter of debate. The PCNA-dependence of this pathway strongly suggested the involvement of either Pol d and/ or e (Frosina et al, 1996) in the resynthesis step. However, the long-patch BER synthesis at a reduced AP site was strongly inhibited by antibodies against Pol b in an in vitro repair assay with cell-free extracts when a linear double-stranded DNA oligonucleotide was used as substrate (Klungland and Lindahl, 1997).…”

“…E cient repair of a uracil-guanine base pair present in a duplex oligonucleotide can be achieved in vitro, via replacement of a single nucleotide (short-patch BER), by the sequential action of the human proteins uracil-DNA glycosylase, the major apurinic/apyrimidinic (AP) endonuclease HAP1, DNA polymerase b (Pol b) and either DNA ligase III (Kubota et al, 1996) or DNA ligase I . The second BER pathway which involves gap-®lling of several nucleotides (longpatch BER) appears to require the same molecular partners with the addition of the proliferating cell nuclear antigen (PCNA) (Frosina et al, 1996) and the structure-speci®c nuclease Flap endonuclease 1 (FEN1) (Klungland and Lindahl, 1997).…”