Interplay of DNA repair pathways controls methylation damage toxicity in Saccharomyces cerevisiae

Abstract

Methylating agents of S(N)1 type are widely used in cancer chemotherapy, but their mode of action is poorly understood. In particular, it is unclear how the primary cytotoxic lesion, O(6)-methylguanine ((Me)G), causes cell death. One hypothesis stipulates that binding of mismatch repair (MMR) proteins to (Me)G/T mispairs arising during DNA replication triggers cell-cycle arrest and cell death. An alternative hypothesis posits that (Me)G cytotoxicity is linked to futile processing of (Me)G-containing base pairs by the MMR system. In this study, we provide compelling genetic evidence in support of the latter hypothesis. Treatment of 4644 deletion mutants of Saccharomyces cerevisiae with the prototypic S(N)1-type methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) identified MMR as the only pathway that sensitizes cells to MNNG. In contrast, homologous recombination (HR), postreplicative repair, DNA helicases, and chromatin maintenance factors protect yeast cells against the cytotoxicity of this chemical. Notably, DNA damage signaling proteins played a protective rather than sensitizing role in the MNNG response. Taken together, this evidence demonstrates that (Me)G-containing lesions in yeast must be processed to be cytotoxic.

Figure 1.—

Experimental set up of the genomewide screen designed to identify factors involved in MNNG-induced ^MeG-DNA damage processing. (A) MNNG-sensitivity of SLP1 (mgt1), SLP15 (mgt1 msh2), SLP19 (mgt1 rad52), and SLP17 (mgt1 msh2 rad52) mutant strains. Graphic representation of the sensitivity at the indicated MNNG concentrations as determined by counting the surviving colonies 48 hr after treatment (left). A representative experiment showing MNNG-treated cells plated at indicated dilutions 24 hr after treatment is shown (right). (B and C) Scheme of genomewide screens for factors contributing to MNNG-sensitivity in the mgt1 rad52 background (B) or required for MNNG-resistance in the mgt1 background (C). Active hypothetical pathways in the bait strain are depicted with bold arrows. See text for details.

Figure 2.—

Mutations influencing cellular response to MNNG and cross-sensitivity to MMS. (A) Factors mediating MNNG sensitivity. SLP19 (mgt1 rad52), sensitive strain; SLP17 (mgt1 rad52 msh2), resistant strain. The genomewide screen and subsequent verification experiments identified exclusively the MMR factors Mlh1, Pms1, Msh2, and Msh6. Approximately 10⁵ cells were treated two times for 1 hr with 0.5 μm MNNG, plated, and evaluated after 24 hr. (B) Cross-sensitivity of MNNG-sensitive deletion mutants to MMS. While the majority of the mutants identified were sensitive to both MNNG and MMS (e.g., rtt101 and cdc50), a few exhibited a significantly differential response. Approximately 10² (left and middle) or 10³ cells (right) were mock treated, treated two times for 1 hr with 3 μm MNNG, or plated on MMS-containing plates and evaluated after 48 hr.

Figure 3.—

Analysis of the msh6 G1067I mutation. (A) CAN1 forward mutation rates of indicated strains. The numbers given are the mutation rates followed by the fold increase relative to wild type in parentheses. (B) Mismatch binding activity in nuclear extracts prepared from wild-type (MSH6), Msh6G1067I (msh6GI), or msh6 null (msh6Δ) cells. The protein/DNA complexes were analyzed by a gel-shift assay and visualized by autoradiography. The specific complex is indicated by an arrow. (C) MNNG-induced killing of strains with indicated genetic backgrounds. Mid-log phase cells were treated with the indicated concentrations of MNNG, spotted at proper serial dilutions on YPD plates, and evaluated after 48 hr as described in materials and methods. The results show a representative experiment.