Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases

Abstract

Despite the progress in understanding the base excision repair (BER) pathway it is still unclear why known mutants deficient in DNA glycosylases that remove oxidised bases are not sensitive to oxidising agents. One of the back-up repair pathways for oxidative DNA damage is the nucleotide incision repair (NIR) pathway initiated by two homologous AP endonucleases: the Nfo protein from Escherichia coli and Apn1 protein from Saccharomyces cerevisiae. These endonucleases nick oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to repair of the remaining 5'-dangling nucleotide. NIR provides an advantage compared to DNA glycosylase-mediated BER, because AP sites, very toxic DNA glycosylase products, do not form. Here, for the first time, we have characterised the substrate specificity of the Apn1 protein towards 5,6-dihydropyrimidine, 5-hydroxy-2'-deoxyuridine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine deoxynucleotide. Detailed kinetic comparisons of Nfo, Apn1 and various DNA glycosylases using different DNA substrates were made. The apparent K(m) and kcat/K(m) values of the reactions suggest that in vitro DNA glycosylase/AP lyase is somewhat more efficient than the AP endonuclease. However, in vivo, using cell-free extracts from paraquat-induced E.coli and from S.cerevisiae, we show that NIR is one of the major pathways for repair of oxidative DNA base damage.

Figure 1

Incision of a supercoiled plasmid DNA by various DNA repair proteins. For the incision assay, 0.8 µg of chemically modified pUC19 and 10 ng of protein were incubated at 37°C for 10 min. The reaction products were separated by electrophoresis on a 0.8% agarose gel in the presence of ethidium bromide (0.5 µg/ml). The gel was photographed and quantified using Bio-Profil software. (A) Relative nicking activity of various enzymes. (B) DMS/alkali-treated plasmid DNA was incubated with different enzymes and analysed by agarose gel electrophoresis. (C) An aliquot of 1 pmol of [3′-³²P]ddAMP-labelled me-FapyGua·C duplex oligonucleotide was incubated with 6 ng of protein at 37°C for 10 min.

Figure 2

Analysis of the substrate specificities of various DNA repair proteins. Activities of Nfo, Apn1, Nth, Fpg and HAP1 proteins towards duplex oligonucleotides containing single DHU, DHT, 5ohU or THF residues. An aliquot of 0.2 pmol of the 5′-³²P-labelled 30mer duplex oligonucleotide was incubated with 3 ng of the given repair proteins at 37°C for 20 min. The reaction products were analysed as described in Materials and Methods.

Figure 3

Base pair specificity of the Nfo (A and B) and Apn1 (C and D) proteins. The initial velocities of cleavage were measured under standard assay conditions and plotted as relative activities. For details see Materials and Methods.

Figure 4

Activities of Nfo, Apn1, Nth and Fpg proteins towards oligonucleotides containing THF, DHU, DHT, me-FapyGua and 5ohU, as a function of time and protein concentration. (A) Time–course plot. Aliquots of 0.6 pmol of the 5′-³²P-labelled oligonucleotides were incubated with 6 ng of a given repair protein at 37°C. (B and C) Enzyme concentration dependence. An aliquot of 1 pmol of the oligonucletide was incubated with the indicated amounts of the repair protein at 37°C for 5 min.

Figure 5

NIR and BER activities in cell-free extracts of E.coli and S.cerevisiae. The [3′-³²P]ddAMP-labelled DHU·G duplex oligonucleotides were incubated with cell-free extract of wild-type and mutant strains of paraquat-induced E.coli (A) or from S.cerevisiae (B) under standard reaction conditions. For details see Materials and Methods.