Apn1 and Apn2 endonucleases prevent accumulation of repair-associated DNA breaks in budding yeast as revealed by direct chromosomal analysis

Abstract

Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).

Figure 1.

BER of alkylation DNA damage in S. cerevisiae [modified from (1)]. An alkylated base is shown as a filled green square. The 5′- and 3′-deoxyribose phosphates (5′-dRP and 3′-dRP) are shown as filled and open blue circles, respectively. Arrowheads on DNA strands correspond to 3'-ends. Red dashed lines represent repair-associated DNA synthesis.

Figure 2.

Measuring chromosome breaks using PFGE in strains containing circular chromosome III. Positions of linearized chromosome III (Chr III) and linear chromosome II (Chr II) are indicated on each gel. (A) Circular chromosome cannot migrate into the gel. ‘L’ – yeast strains with linear chromosome III; ‘Cir’ – yeast strains with circular chromosome III. (B) In vitro treatment of DNA plugs with γ-radiation. Agarose plugs with genomic DNA from G1 haploid cells were placed in buffer (20 mM Tris, pH 8.0 and 50 mM EDTA) and γ -irradiated in the Shepherd Irradiator Model 431 with a 137Cs source at a dose rate of 23 Gy/min. Dose (Gy) is indicated above each lane. Chromosomal DNA in (A) and (B) was visualized by ethidium bromide staining after PFGE. Linearized chromosome III is not visible as a separate band on panel (B); its position is indicated by Southern hybridization. (C) Southern hybridization of the gel shown on panel (B) with a LEU2-probe, which identifies both chromosomes II (Chr II) and III (Chr III).

Figure 3.

PFGE of yeast chromosomes after MMS treatment. The calculated number of chromosome breaks is shown under each lane. (A) MMS-treatment of yeast cells induces heat-labile base damages that can be detected by formation of secondary DSBs. G1 haploid yeast were treated with 0.1% MMS for 15 min (lanes 2, 5, 8, 11) or 30 min (lanes 3, 6, 9, 12) followed by immediate DNA purification and PFGE (lanes 1–3 and 7–9) or PFGE after LH or 24 h in a buffer (LH) (lanes 4–6 and 10–12); lanes 1, 4, 7 and 10 are mock-treated controls. DNA purification with proteinase K digestion was performed either at 55°C (lanes 1–6) to measure heat-dependent breaks (HDBs) or at 30°C (lanes 7–12) to measure heat-independent breaks (HIBs). Chromosomes were visualized by ethidium bromide staining and breaks were quantified based on Southern hybridization. The numbers of DSBs per haploid yeast genome, calculated as described in the text are shown under each lane. (B) MMS treatment of chromosomal DNA within the plug produces heat labile sites. Plugs containing genomic DNA from G1 were treated with 11.8 mM MMS for 15 min (lanes 2, 5, 8) or 30 min (lanes 3, 6, 9); lanes 1, 4 and 7 are mock-treated controls. Lanes 1–3 contain DNA-plugs with no post-treatment incubation; lanes 4–6 contain DNA-plugs that were post-incubated at 30°C for 24 h; lanes 7–9 contain DNA-plugs that were post-incubated at 55°C for 24 h.

Figure 4.

MMS-induced killing of different yeast genotypes used in this study. (A) MMS sensitivity of yeast strains as determined by plating from serial 10-fold dilutions onto rich (YPDA) medium containing 1 mM or 3 mM MMS. (B) Sensitivity of yeast strains treated with 11.8 mM (0.1%) MMS for 15 or 30 min in PBS and then plated to rich (YPDA) medium from serial 10-fold dilutions. (C) MMS sensitivity of G1 yeast or logarithmically growing cells: G1 cells wt (open circle) and mag1 (filled circle); log phase wt (open square) and mag1 (filled square). All experiments were done with two isogenic derivatives of wild-type yeast strains MWJ49 and MWJ50 or two mutant strains derived from these wild types. Plating in (A) and (B) was done 3–4 times for each strain. All numbers in (C) represent the average for two isogenic strains, which differed from each other by no more than 30%.

Figure 5.

A mag1 deletion causes severe blockage in the repair of MMS-induced heat-labile sites. Formation and repair of HDBs (lanes 1–12) and HIBs (lanes 13–24) were assessed as described in the text. Genotypes are indicated. Each group of three lanes (from left to right) contains mock-treated control, 15 min and 30 min MMS-treatment.

Figure 6.

Break accumulation in apn1 apn2 double mutants is modulated by a mag1 deletion. Formation and repair of HIBs (upper panel) and HDBs (lower panel) were assessed as described in the text. Genotypes are indicated above lanes, apn1 apn2 double mutant is abbreviated as apn1/2. Each group of three lanes (from left to right) contains mock-treated control, 15 min and 30 min MMS-treatment.