The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe - PubMed (original) (raw)

Comparative Study

. 2004 Jan 2;32(1):115-26.

doi: 10.1093/nar/gkh151. Print 2004.

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Comparative Study

The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe

Balazs Ribar et al. Nucleic Acids Res. 2004.

Abstract

The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs). Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity. We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sensitizes this fission yeast to alkylation and oxidative damage-inducing agents, which is further enhanced by Apn1 inactivation. We also show that Uve1, present in S.pombe but not in S.cerevisiae, provides the back-up APE activity together with Apn1. We confirmed the presence of APE activity in recombinant Apn2 and in crude cell extracts. Thus S.pombe is distinct from S.cerevisiae, and is similar to mammalian cells in having Apn2 as the major APE.

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Figures

Figure 1

Figure 1

Intron-containing region of the apn1 + (A) and apn2 + (B) genes. The intron sequences are shown in lower case. The consensus sequences for the 5′ splice, branch and 3′ splice sites are boxed [(17) and

http://www.sanger.ac.uk/Projects/S\_pombe

].

Figure 2

Figure 2

(A) Apn1 sequence alignment of S.pombe and S.cerevisiae Apn1with Nfo of E.coli. (B) Apn2 sequence alignment of S.pombe and S.cerevisiae, human APE2, human APE1 and E.coli Xth proteins. Identical and highly conserved residues are indicated in red. Spaces indicate gaps introduced to optimize alignment by the MultiAlin program (23):

http://prodes.toulouse.inra.fr/multalin/multalin.html

. GenBank accession numbers AY483157 for apn1+ and AY483158 for apn2+.

Figure 2

Figure 2

(A) Apn1 sequence alignment of S.pombe and S.cerevisiae Apn1with Nfo of E.coli. (B) Apn2 sequence alignment of S.pombe and S.cerevisiae, human APE2, human APE1 and E.coli Xth proteins. Identical and highly conserved residues are indicated in red. Spaces indicate gaps introduced to optimize alignment by the MultiAlin program (23):

http://prodes.toulouse.inra.fr/multalin/multalin.html

. GenBank accession numbers AY483157 for apn1+ and AY483158 for apn2+.

Figure 3

Figure 3

(A) MMS sensitivity of _apn1_Δ and _apn2_Δ strains. Cells grown overnight in YEL medium were treated with 0.2% MMS for various times. (B) MMS-induced mutagenesis at the can1 gene of S.pombe. The details are described in Materials and Methods. (C) Phleomycin D1 toxicity. Cells grown overnight in YEL medium were treated with phleomycin D1 for 1 h before spreading on YEA plates. (D) UV sensitivity of _S.pombe apn1_Δ and _apn2_Δ mutant strains. Sensitivity of the strains to UV was measured after exposure to various UV doses. PO29, wild type (diamonds); PO61, _apn1_Δ (inverted triangles); PO24, _apn2_Δ (upright triangles); PO60, _apn1_Δ_apn2_Δ (circles).

Figure 4

Figure 4

Effect of _uve1_Δ mutation on MMS sensitivity of the _apn2_Δ mutant. Other details are as in Figure 3. PO29, wild type (diamonds); PO24, _apn2_Δ (triangles); PO89, _uve1_Δ (open circles); PO90, _apn2_Δ_uve1_Δ (open squares).

Figure 5

Figure 5

(A) Complementation of MMS sensitivity of the _apn1_Δ_apn2_Δ (PO60) strain with wild-type S.pombe Apn2-His6 (pNBR110) (filled squares); human APE1 (pRBR113) (filled circles); and human APE2 (pNBR103) (open squares); wild-type control, leu1-32 (PO4), with control CAT expression vector (pNMT1-CAT; Invitrogen) (filled diamonds); sensitivity of _apn1_Δ_apn2_Δ: (PO60) transformed with pNMT1-CAT (open diamonds). Other details are described in Materials and Methods. (B) Agar diffusion assay of complementation of _apn1_Δ_apn2_Δ (PO60) and wild-type (PO4) strains as described in Materials and Methods. Data represent results from an average of three or more experiments. (a) Wild-type Apn2-His6 on plasmid pNBR110 in PO60; (b) control pNMT1-CAT plasmid in PO60; (c) human APE1 on plasmid pRBR113 in PO60; (d) Apn2-A402A403 mutation (PCNA) on plasmid pNBR114 in PO60; (e) Apn2-A456A457A458 mutation on plasmid pNBR115 in PO60; (f) wild-type GST–Apn2 on plasmid pEBR116 in PO60; (g) wild-type Apn1-His6 on plasmid pNBR117 in PO60; (h) pNMT1-CAT plasmid in PO4

Figure 6

Figure 6

(A) Purification of GST–Apn2. Eluted GST–Apn2 fusion protein was analyzed on a 7.5% denaturing polyacrylamide gel and stained with Coomassie blue. Lane 1, molecular weight standards; lane 2, 3 µg of eluted GST–Apn2 protein. (B) Kinetics of APE activity of GST–Apn2; lane (–) is the no-protein control. The details are described in Materials and Methods.

Figure 7

Figure 7

Analysis of APE activity in S.pombe extract. Wild-type and mutant cells were broken by French press and centrifuged. Crude cell extract (1 µg) was mixed with 50 fmol of the oligonucleotide containing the AP site in reaction mixture (20 µl) containing 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT and 100 µg/ml BSA. After incubation at 37°C, the 5′-labeled 30mer product was identified with PhosphorImager (Molecular Dynamics) after electrophoretic separation on 20% polyacrylamide gels containing 7 M urea (26,45).

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