Oncogene (1998) 17, 835 ± 843 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Mammalian base excision repair by DNA polymerases d and e M Stucki1, B Pascucci2, E Parlanti2, P Fortini2, SH Wilson3,UHuÈ bscher1 and E Dogliotti2 1Institut fuÈr VeterinaÈrbiochemie, UniversitaÈtZuÈrich, Winterthurerstrasse 190, 8057 ZuÈrich, Switzerland; 2Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di SanitaÁ, v. le Regina Elena 299, 00161, Roma, Italy; 3Laboratory of Structural Biology, NIEHS, P.O. Box 12233, 11 TW Alexander Drive, Research Triangle Park, North Carolina, 27709, USA Two distinct pathways for completion of base excision structure-speci®c nuclease Flap endonuclease 1 (FEN1) repair (BER) have been discovered in eukaryotes: the (Klungland and Lindahl, 1997). DNA polymerase b (Pol b )-dependent short-patch path- Several studies have attempted to address the issue way that involves the replacement of a single nucleotide of the identity of the Pols involved in the DNA and the long-patch pathway that entails the resynthesis synthesis step that ®ll in gaps created during repair. Pol of 2-6 nucleotides and requires PCNA. We have used cell d and/or Pol e have been implicated in the synthesis extracts from Pol b-deleted mouse ®broblasts to separate step of the other two major excision repair processes, subfractions containing either Pol d or Pol e. These nucleotide excision repair (Shivji et al., 1995) and fractions were then tested for their ability to perform mismatch repair (Longley et al., 1997) which both both short- and long-patch BER in an in vitro repair require PCNA (Shivji et al., 1992, Umar et al., 1996). assay, using a circular DNA template, containing a As far as BER is concerned the characteristics of single abasic site at a de®ned position. Remarkably, both mouse ®broblasts cell lines with a homozygous deletion Pol d and Pol e were able to replace a single nucleotide of Pol b (Sobol et al., 1996) give the most clear at the lesion site, but the repair reaction is delayed evidence that Pol b functions speci®cally in this compared to single nucleotide replacement by Pol b. pathway in the replacement of a single damaged Furthermore, our observations indicated, that either Pol nucleotide. In contrast, however, the identity of the d and/or Pol e participate in the long-patch BER. PCNA Pols involved in the long-patch BER is still matter and RF-C, but not RP-A are required for this process. of debate. The PCNA-dependence of this pathway Our data show for the ®rst time that Pol d and/or Pol e strongly suggested the involvement of either Pol d and/ are directly involved in the long-patch BER of abasic or e (Frosina et al., 1996) in the resynthesis step. sites and might function as back-up system for Pol b in However, the long-patch BER synthesis at a reduced one-gap ®lling reactions. AP site was strongly inhibited by antibodies against Pol b in an in vitro repair assay with cell-free extracts Keywords: DNA repair; DNA polymerases; abasic sites when a linear double-stranded DNA oligonucleotide was used as substrate (Klungland and Lindahl, 1997). In order to clarify this matter we have used whole cell extracts from Pol b-deleted mouse ®broblasts to Introduction separate subfractions containing either Pol d or Pol e. These fractions were then tested for their ability to The integrity of DNA in vivo is continuously perform both short-and long-patch BER in an in vitro challenged by a variety of exogenous and endogenous repair assay. Here, we show that either Pol d and/or agents as well as by cellular processes. Among the Pol e participate in long-patch BER and might defence mechanisms that cells have evolved to counter- function as back-up systems for Pol b in one-gap act these threats, the base excision repair (BER) ®lling reactions. Moreover, the role played by the pathway is the main strategy to correct both replication auxiliary proteins PCNA, replication spontaneous DNA damage and small DNA adducts. protein A (RP-A) and replication factor C (RF-C) in This repair system involves the removal of a base and BER is addressed. its replacement by a chain of enzymatic steps. Ecient repair of a uracil-guanine base pair present in a duplex oligonucleotide can be achieved in vitro, via replace- Results ment of a single nucleotide (short-patch BER), by the sequential action of the human proteins uracil- DNA Characterization of the Pol d and Pol e-containing glycosylase, the major apurinic/apyrimidinic (AP) fractions endonuclease HAP1, DNA polymerase b (Pol b)and either DNA ligase III (Kubota et al., 1996) or DNA Whole cell extracts from Pol b-deleted mouse ®broblasts ligase I (Prasad et al., 1996). The second BER pathway (Sobol et al., 1996) were fractionated to separate Pol d which involves gap-®lling of several nucleotides (long- and Pol e. In a ®rst fractionation step over phospho- patch BER) appears to require the same molecular cellulose, the extract was depleted from PCNA and RP- partners with the addition of the proliferating cell A. The second fractionation step over hydroxylapatite nuclear antigen (PCNA) (Frosina et al., 1996) and the led to a separation of Pol a from Pol d/e. The three fractions obtained after the third step over Mono Q, PFIIA, B and C, were analysed in more detail (Figure Correspondence: E Dogliotti 1). Poly(dA)oligo(dT) was used in combination with BP and EP contributed equally to this work Received 19 January 1998; revised 25 March 1998; accepted 26 PCNA to distinguish between Pol d- and Pol e- March 1998 containing fractions, since Pol d is inactive in this DNA polymerase d and e in base excision repair M Stucki et al 836 assay without PCNA (Weiser et al., 1991). As shown in activity of PFIIB and PFIIC was unmodi®ed in the Figure 1a, in the presence of PCNA (open circles) all presence of the Pol a-speci®c neutralizing antibody SJK three fractions were able to substain DNA synthesis, 132-20 and the absence of Pol a in these two fractions while in the absence of PCNA (closed triangles) only was also con®rmed by immunoblot analysis (data not PFIIB showed DNA synthesis activity. Fractions PFIIB shown). PFIIB and PFIIC were then named PFIIe and and PFIIA/C were therefore good candidates for Pol e- PFIId, respectively. Further immunoblot analysis and Pol d-containing fractions, respectively. Immuno- revealed that PFIIe and PFIId contained in addition blot analysis (Figure 1b) con®rmed that PFIIC DNA ligase I and replication factor C (RF-C) (Figure contained Pol d (lane 2, left side) but no Pol e (lane 2, 1c, lanes 2 and 3, respectively) but were free of PCNA right side), while PFIIB contained Pol e (lane 3, right and RP-A (data not shown). side) and was free of Pol d (lane 3, left side). Immunoblot analysis of PFIIA revealed that this Pol d and Pol e can perform short-patch base excision fraction contained another form of Pol d, with a large repair subunit migrating at approximately 130 kDa (data not shown). This fraction was not further analysed. The The experimental system used in this study (Figure 2) total level of PCNA-dependent and -independent to monitor BER involves the use of a circular duplex Figure 1 Ion-exchange chromatography and immunological characterization of the Pol-containing fractions from Pol b-null cell extract. Chromatography was performed as described under Materials and methods. (a) Activity pro®le. Fractions were tested for activity as described under Material and methods; open circles, PCNA- dependent Pol activity, closed triangles, PCNA-independent Pol activity. PFIIB and PFIIC eluted at *260 and *430 mM potassium phosphate, respectively. (b) Immunoblot analysis of total cell extract and the MonoQ fractions PFIIB and PFIIC with antibodies against Pol d (left side) and Pol e (right side). Lane 1: 10 mg total cell extract; lane 2: 3 mg PFIIC; lane 3: 3 mg PFIIB. (c) Immunoblot analysis of total cell extract and the MonoQ fractions PFIIB and C with antibodies against DNA ligase I (left side) and RF-C (right side). Lane 1: 10 mg total cell extract; lane 2: 3 mg PFIIB (peak and side fractions); lane 3: 3 mg PFIIC (peak and side fractions). Gel electrophoresis and immunoblot analysis were performed as described in Materials and methods DNA polymerase d and e in base excision repair M Stucki et al 837 plasmid molecule containing a single abasic site (X) required for the long-patch but not for the short-patch and of dierent labeled dNTPs in the reaction mixture BER (Frosina et al., 1996; Fortini et al., 1998) the in order to distinguish between short- and long-patch dependence of the repair reaction on this auxiliary repair events. The incorporation of radiolabeled dTMP protein is an additional marker to discriminate between in the BamHI ± HindIII restriction fragment marks the two pathways. mainly the occurrence of one-gap ®lling reactions that First, the ability of these two fractions to perform are the major route for repair of AP sites, while the short-patch BER was monitored as a function of time incorporation of dCMP identi®es exclusively 2-6 (Figure 3). Repair products were analysed by nucleotides repair patches. Moreover since PCNA is autoradiography after electrophoretic separation in denaturing 15% PAGE. Three labeled DNA products were visible on the autoradiography: the internal standard (IS) which is a 5'end-labeled 60-mer added in all reaction tubes in order to correct the repair incorporation values (as measured by electronic autoradiography, Figure 3 right) for DNA recovery; the 30-mer which is a marker of fully repaired DNA and the 13-mer which is the repair intermediate arising from one-nucleotide replacement reactions.