A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

Abstract

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1.

Figure 1

MMS sensitivity of apn2 and nth1 single and double mutants. S.pombe wild-type (closed diamonds), apn2 (closed squares), nth1 (closed triangles), and apn2, nth1 (crosses) mutant cells were plated on solid media containing increasing doses of MMS and colony survival calculated relative to plates without MMS.

Figure 2

Spontaneous canavanine forward mutations in cells lacking apn2 and nth1. Logarithmically growing S.pombe cells were plated on EMM plates supplemented with l-canavanine sulfate for the determination of spontaneous mutation frequencies to Can1R in wild-type (WT), apn2, nth1 and apn2, nth1 mutant cells.

Figure 3

DNA incision and borohydride protein complex trapping at AP sites by cell extracts from wild–type (WT) and nth1 mutant cells. (A) Cleavage of AP DNA in S.pombe cell-free extracts. Duplex 24mer oligodeoxyribonucleotide containing a single abasic site (opposite G) was incubated with 0.1, 0.4 or 1 μg of whole cell extracts from wild-type (FO656) and nth1 mutant (FO763) cells in reaction buffer supplemented with 5 mM MgCl₂. Cleaved DNA was separated from intact DNA using PAGE and visualized by phosphorimaging. Purified Apn1 (15 ng) from S.cerevisiae and Nth1 (15 ng) from S.pombe were used as markers of AP endonuclease and AP lyase cleavage, respectively. (B) Probing for covalent enzyme–AP DNA intermediates by NaCNBH₃ reduction. An aliquot of 2.5 or 10 μg S.pombe whole cell extract, 20 ng E.coli Fpg or no enzyme was incubated with 10 fmol of a 24mer DNA duplex containing a single AP site opposite C in the presence of 50 mM NaCNBH₃. Protein–DNA complexes were separated from DNA by SDS–PAGE.

Figure 4

DNA glycosylase activities in cell extracts from S.pombe. (A) Cleavage of A/8oxoG-containing duplex DNA. An aliquot of 0.5 and 2.5 μg of protein extract from wild-type or nth1 cells, 20 ng MutY from E.coli and 10 ng purified Apn1 from S.cerevisiae, or 10 ng Apn1 only, were incubated with 10 fmol of a 24mer³²P-labelled oligodeoxyribonucleotide harbouring an A opposite 8oxoG in the presence of 5 mM Mg²⁺. Strand cleavage was analysed by 20% denaturing PAGE and phosphorimaging. (B) MutY activity in wild-type and nth1 extracts. Protein extracts (2.5 µg) from wild-type or nth1 cells were incubated with A:8oxoG DNA (as in A) and subsequently treated with 100 mM NaOH. Relative cleavage was quantified with the ImageQuaNT software. (C) Uracil removing and nicking activity in wild-type protein extracts of S.pombe. Whole cell extract (0.5 μg) was incubated with 10 fmol of a 24 bp oligodeoxyribonucleotide containing a single uracil residue (opposite A) at position 14 with or without S.pombe Nth1 (2, 5 or 10 ng) or S.cerevisiae Apn1 (100, 250 or 500 pg) for 30 min at 37°C. Similar experiments with Udg (NEB) were also included as indicated. The reaction products were separated on a polyacrylamide gel and bands were detected by phosphorimaging.