Different DNA Polymerases Are Involved in the Short- and Long-Patch Base Excision Repair in Mammalian Cells

Abstract

Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.

Mammalian base excision repair by DNA polymerases δ and ε

“…Whole cell extracts from Pol b-null cell lines (Fortini et al, 1998) as well as fractionated cell extracts containing either Pol d or Pol e (this study) were able to perform both the short-and the longpatch BER. Typically an abasic site is recognized by an AP endonuclease that incises the phosphodiester backbone immediately 5' to the lesion leaving a strand break with a 5'-abasic terminus.…”

“…In light of these results and of a recent own study (Fortini et al, 1998) we wish to reconsider the model proposed for BER in mammalian cells (Klungland and Lindahl, 1997). Whole cell extracts from Pol b-null cell lines (Fortini et al, 1998) as well as fractionated cell extracts containing either Pol d or Pol e (this study) were able to perform both the short-and the longpatch BER.…”

“…The addition of a b-catalyst like spermine is in fact required to complete short-patch BER by PFIId and PFIIe. The accumulation of repair intermediates was not observed when cell extracts from Pol b-deleted cells were used in the short-patch BER reaction (Fortini et al, 1998). It is possible that molecules able to act as b-elimination catalysts were lost in the fractionation procedure.…”

“…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.…”