The activity of yeast Apn2 AP endonuclease at uracil-derived AP sites is dependent on the major carbon source

Abstract

Yeast Apn2 is an AP endonuclease and DNA 3'-diesterase that belongs to the Exo III family with homology to the E. coli exonuclease III, Schizosaccharomyces pombe eth1, and human AP endonucleases APEX1 and APEX2. In the absence of Apn1, the major AP endonuclease in yeast, Apn2 can cleave the DNA backbone at an AP lesion initiating the base excision repair pathway. To study the role and relative contribution of Apn2, we took advantage of a reporter system that was previously used to delineate how uracil-derived AP sites are repaired. At this reporter, disruption of the Apn1-initiated base excision repair pathway led to a significant elevation of A:T to C:G transversions. Here we show that such highly elevated A:T to C:G transversion mutations associated with uracil residues in DNA are abolished when apn1∆ yeast cells are grown in glucose as the primary carbon source. We also show that the disruption of Apn2, either by the complete gene deletion or by the mutation of a catalytic residue, results in a similarly reduced rate of the uracil-associated mutations. Overall, our results indicate that Apn2 activity is regulated by the glucose repression pathway in yeast.

Keywords: AP endonuclease; DNA repair; Glucose repression; Uracil in DNA.

Fig. 1

The rates of Lys+ and Can^R mutations in various carbon sources. In all graphs, error bars indicate 95% confidence intervals. Two rates are considered significantly different when the error bars do not overlap. a Rates of Lys+ mutations at pTET-lys2-TAA in the indicated strain backgrounds. Rates were measured by fluctuation analyses in cells cultured in YEP media supplemented with either 2% Glycerol+ 2% ethanol (YEPGE) or 2% glucose (YEPD). b Rates of canavanine-resistant mutations at pTET-lys2-TAA in the indicated strain backgrounds. Rates were measured by fluctuation analyses in cells cultured in YEP media supplemented with either 2% glycerol+ 2% ethanol (YEPGE) or 2% glucose (YEPD). c Rates of Lys+ mutations at pTET-lys2-TAA in the indicated strain backgrounds. Rates were measured by fluctuation analyses in cells cultured in YEP media supplemented with either 2% glycerol or 2% raffinose

Fig. 2

The requirement of Apn2 catalytic activity for the elevated Lys+ mutations in glycerol/ethanol. In all graphs, error bars indicate 95% confidence intervals. Two rates are considered significantly different when the error bars do not overlap. a Rates of Lys+ mutations at pTET-lys2-TAA in either apn1Δ apn2Δ (YNK103; see Table S1) or apn2Δ apn1Δ (YNK219; see Table S1). Rates were measured by fluctuation analyses in cells cultured in YEP media supplemented with either 2% glycerol+ 2% ethanol (YEPGE) or 2% glucose (YEPD). b Rates of Lys+ mutations at pTET-lys2-TAA in YNK103 (apn1Δ::loxP apn2Δ::loxP-TRP1-loxP) transformed with either the vector (pGPD2; Addgene #43,972) or pNK229 (pGPD-APN2). Rates were measured by fluctuation analyses in cells cultured in synthetic, uracil-deficient medium (SC-URA) containing either 2% glucose or 2% glycerol+ 2% ethanol as indicated. c Rates of Lys+ mutations at pTET-lys2-TAA in YNK98 (apn1Δ APN2), YNK103 (apn1Δ apn2Δ), or YNK658 (apn1Δ apn2E59A). Rates were measured by fluctuation analyses in cells cultured in YEP media supplemented with either 2% Glycerol+ 2% ethanol (YEPGE) or 2% glucose (YEPD)

Fig. 3

Stress response pathways and the elevated Lys+ mutations in glycerol/ethanol. In all graphs, error bars indicate 95% confidence intervals. Two rates are considered significantly different when the error bars do not overlap. a–h Rates of Lys+ mutations at pTET-lys2-TAA in the indicated strain backgrounds. Rates were measured by fluctuation analyses in cells cultured in YEP media supplemented with either 2% Glycerol + 2% ethanol (YEPGE) or 2% glucose (YEPD) as indicated above each graph. For more information about the strains used, see Table S1

Fig. 4

Apn2 protein level in WT, apn1Δ, and apn1Δ rfx1Δ backgrounds. Western blot analysis of yeast whole cell extracts in the indicated strain backgrounds. C-terminally flag-tagged Apn2 was detected by anti-flag antibody. Anti-GAPDH was used as loading control. The locations of relevant molecular weight marker bands are indicated at the right of western blot

Fig. 5

Putative post-translational modification sites of Apn2. The protein sequence of S. cerevisiae Apn2 is shown in the box with the catalytic glutamate 59, putative Sumoylation sites (GPS-SUMO Webserver: http://sumosp.biocuckoo.org), and putative ubiquitination sites (UBpred: http://www.ubpred.org) indicated in red, green, and blue, respectively. Underlined residues (IVIIS) are predicted to be SUMO-interaction site (GPS-SUMO webserver: http://sumosp.biocuckoo.org)