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. Author manuscript; available in PMC: 2011 Apr 4.
Published in final edited form as: DNA Repair (Amst). 2010 Feb 4;9(4):458–467. doi: 10.1016/j.dnarep.2010.01.009

RAD51D protects against MLH1-dependent cytotoxic responses to O6-methylguanine

Preeti Rajesh 1, Changanamkandath Rajesh 1, Michael D Wyatt 1,*, Douglas L Pittman 1,*
PMCID: PMC2858319  NIHMSID: NIHMS177831  PMID: 20133210

Abstract

SN1-type methylating agents generate O6-methyl guanine (O6-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O6-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR dependent mutagenicity and toxicity caused by O6-meG has been studied, the mechanisms of recombination induced by O6-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d−/−Mlh1−/− Trp53−/− MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3 fold sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of γ-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing γ-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O6-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O6-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O6-meG adducts.

Keywords: Alkylating agents, Homologous recombination, Mismatch repair, MLH1, RAD51D

1. Introduction

SN1-type methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) are potent genotoxic agents that produce a spectrum of methyl DNA adducts. O6-methylguanine (O6-meG), which constitutes only 8–10% of the total adducts, is extremely mutagenic because it mispairs with thymine during DNA replication, reviewed in [1–3]. O6-meG is also highly clastogenic and cytotoxic, although the precise sources of these effects have not been fully elucidated. The cytotoxicity of O6-meG is exploited with clinically used methylating agents such as dacarbazine, streptozotocin, procarbazine, and temozolomide [1].

The first line of defense against O6-meG damage is the direct reversal of the methyl adduct by O6-methylguanine methyltransferase (MGMT). The methyl groups are removed in a suicide reaction that inactivates the protein [2]. MGMT levels vary widely among individuals, and expression of this gene is susceptible to epigenetic silencing [4,5]. Therefore, other DNA repair pathways are frequently involved in the cellular response to this adduct. O6-meG readily mispairs with thymine during DNA replication and is recognized by mismatch repair (MMR), but this recognition results in cell death instead of repair. In fact, MMR appears to be the only pathway in S. cerevisiae that sensitizes cells to MNNG [6]. Loss of any one of four mammalian MMR components, namely MSH2:MSH6 (MutSα) and MLH1:PMS2 (MutLα), renders cells dramatically resistant to killing caused by O6-meG [7].

How MMR contributes to the cytotoxicity of O6-meG is a subject of debate. One model ascribes O6-meG-mediated lethality to repetitive and futile attempts of MMR to process the O6-meG:T mismatches [8]. Another model suggests that MutSα and MutLα-dependent recognition of O6-meG:T mismatches directly triggers DNA damage checkpoint signaling [9]. ATM and ATR are central signaling nodes activated by DNA damage and replication-associated stress that subsequently coordinate the activation of cell cycle checkpoint responses. MutSα and MutLα binding to O6-meG:T activates the ATR/CHK1 signaling cascade [9–11]. Regardless of the model, recognition of O6-meG:T mismatches by MMR proteins results in a potent signal of apoptosis [12,13]. Furthermore, p53 is not absolutely required for MMR dependent apoptosis triggered by O6-meG [14,15].

Unlike MMR, the homologous recombination (HR) DNA repair pathway protects against the cellular lethality caused by methylating agents [16–18]. MNNG induces sister chromatid exchanges (SCEs), which are dependent upon MutSα and MutLα [19–23]. Loss of MSH2 or MLH1 alleviates the induction of SCEs in addition to the cytotoxicity, suggesting that the HR events protect against lethality. MMR-dependent recognition of O6-meG:T is hypothesized to cause a DNA strand break event that must be resolved by HR [18]. However, it is unknown exactly what DNA break intermediate is created by MMR recognition of O6-meG:T, and the precise role of HR in this regard remains poorly understood.

RAD51D is a member of the RAD51 protein family that performs central roles in HR repair. RAD51D is essential for normal development and cellular proliferation [24,25]. Previously, we demonstrated that absence of p53 partially bypasses the embryo-lethal phenotype conferred by a Rad51d deletion in mice and generated Rad51d Trp53-deficient MEFs [25]. These HR-deficient cells had extensive chromosomal instability and were hypersensitive to DNA damaging agents including methyl methanesulfonate (MMS), an SN2-type methylating agent that causes N-methylpurine adducts but does not measurably cause O6-meG. In this study, we examined the role of RAD51D in response to the SN1-type methylating agent MNNG. We show that Rad51d-deficient MEFs are hypersensitive to MNNG. Moreover, deletion of the MMR gene Mlh1 partially rescued the sensitivity of Rad51d-deficient cells. This was accomplished by crossing MMR and HR-deficient mice to generate lines heterozygous for Mlh1, Rad51d, and Trp53. Absence of Mlh1 does not appear to affect the embryo lethal phenotype of Rad51d-deficient and Rad51d Trp53-deficient embryos. In the Rad51d Trp53-deficient MEFs, the G2/M arrest and cell death is substantially worsened, while loss of MMR dramatically alleviates these phenotypes. Collectively, the data conclusively demonstrate that RAD51D-mediated HR protects against lethality induced by MMR processing of O6-meG lesions.

2. Materials and methods

2.1. Breeding Scheme, Genotyping and Generation of Cell Lines

Mice heterozygous for Rad51d [24,25], Rad51d Trp53 [25] and Mlh1 null alleles [26] were crossed to generate offspring heterozygous for Rad51d Mlh1, Rad51d Trp53 Mlh1 and Mlh1 Trp53 genes. Mlh1 mice were provided by the COBRE Mouse Core Facility at the University of South Carolina. All three lines were maintained in a C57BL6/J strain background. Following timed matings, embryos from E9.5 to E15.5 were dissected and either fixed in 4% paraformaldehyde or homogenized for generating Mouse Embryonic Fibroblast (MEF) cell lines [25]. Rad51d, Trp53 and Mlh1 genotypes of embryos were determined as described [25,26]. All animal procedures were performed in compliance with federal and institutional guidelines.

2.2. Colony Survival Assays

MEFs were seeded onto 100-mm dishes at 1000 cells per plate. For colony survival assays, DMEM supplemented with 10% Fetal Bovine Serum, Glutamine and antibiotics was used. Cells were treated with MNNG (Sigma-Aldrich, MO, USA) or MMS (Sigma) at the indicated concentrations. For experiments involving MNNG, because of the short half-life of MNNG (1 h), the medium was not replaced. For experiments involving MMS, cells were treated with MMS for 1 h and the medium was replaced. During the course of the experiment, the medium was replaced once after 5 days. Following treatments, cells were grown for 8 to 10 days, fixed with 100% methanol and stained with Giemsa (Sigma-Aldrich, MO, USA) [25]. Colonies containing 50 or more cells were counted. Three independent experiments were performed for each agent and each experiment performed in triplicates. The error bars represent the standard error of means. The fold differences were calculated from dose required for 50% cell death.

2.3. Cell Cycle Analysis by Flow Cytometry

For analysis of cell cycle responses upon exposure to MNNG, 1.0 × 105 cells were seeded onto 100 mm dishes. 48 h later medium containing 1 µM MNNG was added to the treatment plates, while medium without the drug was added to control plates. At the indicated time points, all cells were pelleted following trypsinization and gently resuspended in propidium iodide (50 µg/ml) staining solution containing 4 mM sodium citrate (pH 7.8), 30 units/ml RNAse (Sigma), and 0.1% Triton X-100. After an incubation period of 10 min at 37° C, NaCl was added to a final concentration of 0.15 M [27]. Cell cycle analyses were performed using a Beckman Coulter FC 500 cytometer (Beckman Coulter, CA, USA) and data quantified using ModFit LT software version 3.1 (Verity Software House, ME, USA).

2.4. Caspase-3 Activation Assay

The activity of caspase-3 (CPP32/apopain) was determined by the EnzChek Caspase-3 Assay Kit number 2 (Invitrogen, CA, USA) following manufacturer’s protocol. Briefly, 5 × 105 cells were seeded on 100 mm plates. Cells were either treated with 1 µM MNNG or left untreated (control). Both treated and control cells were then harvested at the indicated time points, washed in phosphate-buffered saline (PBS) and cell pellets were lysed and assayed as described in the kit protocol. Reactions were carried out at room temperature and fluorescence was measured by DTX880 fluorescence plate reader (Beckman Coulter) using excitation at 485nm and emission at 530 nm. The fluorescence data was normalized to 106 cell count per reaction.

2.5. Western Blot Analysis

MEF cell line of each genotype was treated with 1 µM MNNG for indicated time points. Whole cell protein extract was prepared from each cell line using M-PER extraction reagent (Thermo Scientific, IL, USA) containing Complete™ mini protease inhibitor cocktail (Roche Life Sciences, NJ, USA) and PhosSTOP phosphatase inhibitor cocktail (Roche). 60 µg of the cell lysates were resolved on SDS-polyacrylamide gel, and transferred onto nitrocellulose membrane (GE healthcare, NJ, USA). The blots were blocked for 1 hour using 5% nonfat skim milk in 20 mM Tris buffered saline containing 0.1% (v/v) Tween-20 (TBST). The blots were then incubated with diluted primary antibody overnight at 4° C. Primary antibodies used in this study include 1:1000 rabbit polyclonal anti-γ-H2AX (Trevigen, MD, USA), 1:1000 mouse monoclonal anti-CHK1, 1:1000 rabbit monoclonal anti-p-CHK1 (Ser345), 1:1000 rabbit monoclonal anti-ATM (Cell Signaling, MA, USA), 1:1000 rabbit polyclonal anti-p-ATM (Ser 1987) (R & D systems, MN, USA) and 1:5000 mouse monoclonal anti-β-actin (Abcam, MA, USA). 1:5000 dilution of the respective horseradish peroxidase-conjugated secondary antibodies (Santa Cruz biotechnology, CA, USA; Abcam) were used followed by detection by west pico chemiluminescent reagent (Thermo Scientific). For MGMT protein expression analysis, 15 µg of the cell lysates of each MEF cell line were similarly immunoblotted using 1:1000 rat monoclonal anti-MGMT primary antibody (R & D systems, MN, USA).

2.6. Statistical analysis

Calculations of the mean, standard deviation and standard error were performed using Microsoft Excel. Statistical analysis for comparison of each set of experimental means was performed using Graphpad Instat 3.0 (Graphpad Software Inc., La Jolla, CA). Significance of data was determined by paired t-test. A value of P <0.05 was considered statistically significant.

3. Results

3.1. Absence of Mlh1 does not affect the embryo lethal phenotype of Rad51d-deficient embryos

HR inactivation hypersensitizes S. cerevisiae to MNNG and defects in the MMR genes Mlh1 and Msh2 rescue this sensitivity [6]. To collectively explore MMR and HR in a mammalian system, Rad51d+/− and Mlh1+/− mice were crossed to generate offspring heterozygous at both loci. Rad51d+/− Mlh1+/− mice were then intercrossed and embryos dissected to determine whether the absence of Mlh1 alters the embryo lethality conferred by a Rad51d deletion. Expected genotypic ratios were observed up to 11.5 dpc (Table 1). No phenotypic differences between Rad51d−/− and Rad51d−/− Mlh1−/− embryos were observed and lethality occurred at the same stage (Fig. 1A). These results suggest that MMR deficiency does not alter the fate of Rad51d defects during embryo development. To further verify this conclusion, Rad51d+/− Trp53+/− and Mlh1+/− mice were crossed to generate offspring heterozygous at all three loci and intercrosses performed. Expected genotypic ratios were observed up to day 15.5 dpc (Table 2). Rad51d−/− Trp53−/− and Rad51d−/− Trp53−/− Mlh1−/− embryo phenotypes were similar with regards to gestational progression and overt appearance, consistent with the phenotype observed in the Rad51d and Mlh1 crosses performed in a p53 proficient background (Fig. 1B). These observations indicate that Mlh1 deficiency does not appreciably influence the developmental phenotypes conferred by the absence of Rad51d during early and late stage gestation. This was an important first step in the study because, to our knowledge, this is the first characterization of a cross between MMR and HR deficient animals.

Table 1.

Genotypes of progeny from Rad51d+/−Mlh1+/− intercrosses

Expected Genotypes Observed (expected)
9.5dpc 10.5dpc 11.5dpc
Rad51d+/+Mlh1+/+ 0 (2.1) 1 (3.1) 3 (1.6)
Rad51d+/+Mlh1+/− 6 (4.3) 7 (6.1) 2 (3.1)
Rad51d+/−Mlh1+/+ 5 (4.3) 8 (6.1) 7 (3.1)
Rad51d+/−Mlh1+/− 9 (8.5) 13 (12.3) 4 (6.3)
Rad51d+/+Mlh1−/− 2 (2.1) 4 (3.1) 3 (1.6)
Rad51d+/−Mlh1−/− 3 (4.3) 8 (6.1) 2 (3.1)
Rad51d−/−Mlh1+/+ 2 (2.1) 3 (3.1) 1 (1.6)
Rad51d−/−Mlh1+/− 5 (4.3) 4 (6.1) 1 (3.1)
Rad51d−/−Mlh1−/− 2 (2.1) 1 (3.1) 2 (1.6)
Total 34 49 25
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Fig. 1.

Fig. 1

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An Mlh1 deletion does not affect the phenotype of Rad51d null embryos. (A & B) Representative E11.5 (A) and E15.5 (B) embryos in an order of wild-type, Rad51d−/−Mlh1−/− and Rad51d−/−Mlh1−/− in Trp53+/+ and Trp53−/− background respectively. (C) Genotypes of the embryos as determined by PCR. For the Rad51d gene, 285- and 250-bp fragments identify the disrupted and wild-type alleles, respectively. For the Trp53 gene, 600- and 450-bp fragments identify the disrupted and wild-type alleles, respectively. For the Mlh1 gene, 198- and 258-bp fragments identify the disrupted and wild-type alleles, respectively. (D) MGMT protein expression was analyzed in Mlh1−/−Trp53−/−, Rad51d−/−Trp53−/−Mlh1−/−, Rad51d−/−Trp53−/−, Trp53−/−, and wild-type MEFs by Western blot. β-Actin was used as a loading control.

Table 2.

Genotypes of progeny from Rad51d+/−Trp53+/−Mlh1+/− intercrosses

Expected Genotypes Observed (expected)
12.5dpc 14.5dpc/15.5dpc
Rad51d+/+Trp53+/+Mlh1+/+ 1 (2.1) 4 (2.6)
Rad51d+/+Trp53+/+Mlh1+/− 3 (4.3) 11 (5.3)
Rad51d+/−Trp53+/−Mlh1+/+ 5 (4.3) 8 (5.3)
Rad51d+/−Trp53+/−Mlh1+/− 8 (8.5) 6 (10.5)
Rad51d+/+Trp53+/+Mlh1−/− 3 (2.1) 2 (2.6)
Rad51d+/−Trp53+/−Mlh1−/− 4 (4.3) 5 (5.3)
Rad51d−/−Trp53−/−Mlh1+/+ 4 (2.1) 2 (2.6)
Rad51d−/−Trp53−/−Mlh1+/− 2 (4.3) 2 (5.3)
Rad51d−/−Trp53−/−Mlh1−/− 4 (2.1) 2 (2.6)
Total 34 42
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3.2. RAD51D functions downstream of MLH1 to protect against lethality induced by MNNG

To characterize the genetic interactions between HR and MMR pathways in response to SN1-type alkylation base damage, MEF cell lines null for both Rad51d and Mlh1 were derived from embryos of Mlh1+/−Rad51d+/−Trp53+/− intercrosses. Because Rad51d−/− MEFs with wild-type p53 fail to proliferate, comparisons between MLH1 and RAD51D status were made with Trp53−/− cells. The genotypes of the embryos were verified by genomic PCR (Fig. 1C). Four independent Mlh1−/−Rad51d−/−Trp53−/− triple knockout cell lines, two Mlh1−/−Trp53−/− cell lines, and numerous cell lines of each heterozygous genotype combination were generated for subsequent analysis.

Because MGMT can directly remove O6-meG, MGMT expression in each MEF line was examined at the RNA and protein levels. Fig. 1D shows nearly undetectable MGMT protein in all Trp53-deficient MEFs regardless of Mlh1 or Rad51d genotypes. In contrast, a triple wild-type (Mlh1+/+ Trp53+/+ Rad51d+/+) cell line expressed substantial levels of MGMT. Analysis of mRNA by real-time-PCR (not shown) demonstrated that the loss of MGMT expression occurred at the level of transcription. Importantly, these results showed that MGMT activity is not a factor for the cellular responses in this model system.

To evaluate RAD51D/MLH1 epistasis in the response to O6-meG adducts, colony survival assays were performed using MEFs of all four genotypes (Trp53−/−, Rad51d−/− Trp53−/−, Mlh1−/−Trp53−/−, and Rad51d−/− Mlh1−/− Trp53−/−) following treatment with MNNG across a range of doses. The Rad51d−/− Trp53−/− MEFs were sensitized to MNNG by 5.3-fold compared to the Rad51d+/+ Trp53−/− MEFs (Fig. 2A). Note that the cell lines utilized in Fig. 2A are both Mlh1+/+. The results were verified in two independent Rad51d−/− Trp53−/− and Rad51d+/+ Trp53−/− MEF cell lines, and MLH1 protein expression was confirmed by western blot (data not shown). Fig. 2B shows that MNNG sensitivity is dependent upon Mlh1 status in Rad51d+/+ cells. The Mlh1−/− Trp53−/− MEFs were dramatically resistant to MNNG compared to Mlh1+/+ Trp53−/− MEFs, confirming that loss of MutSα/MutLα renders cells resistant to methylating agents that produce O6-meG. The sensitivity of triple knockout (Rad51d−/− Mlh1−/− Trp53−/−) MEFs to MNNG was examined to determine what happens when MMR is lost in HR-deficient cells. The data in Fig. 2C conclusively demonstrate that loss of MMR indeed alleviated sensitivity of Rad51d-deficient cells to MNNG by 5.2 fold compared to Rad51d−/− Trp53−/− MEFs (Fig. 2C). The observation was verified in two independent triple knockout MEF cell lines (not shown).

Fig. 2.

Fig. 2

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RAD51D is required for resistance to O6-meG lesions in MMR-proficient MEFs. Colony survival assays of MEFs deficient in Rad51d, Mlh1 or both, treated with MNNG at the indicated doses. (A) Pair-wise comparison of Rad51d-proficient (♦) and Rad51d-deficient (■) MEFs. (B) Pair-wise comparison of Mlh1-proficient (♦) and Mlh1-deficient MEF (○). (C) Pair-wise comparison of Rad51d-deficient Mlh1-proficient (■) and Rad51d-deficient Mlh1-deficient (▲) MEFs. Clonogenic survival assays show that deleting Mlh1 in Rad51d-deficient cells rescues the sensitivity of these cells to MNNG 5.2-fold. Survival is plotted as percentage compared to untreated control. Each data point represents the mean of three independent experiments each performed in triplicates. Error bars represent the standard error of means. All MEFs are Trp53−/−.

Loss of Mlh1 substantially alleviated (5.2-fold), but did not completely rescue the MNNG-sensitive phenotype of the Rad51d−/− MEFs (compare ○ in Fig. 2B to ▲ in Fig. 2C). Like MMS, MNNG produces a large proportion of 7-methylguanine, and Rad51d−/− MEFs are sensitive to MMS [25]. To substantiate the hypothesis that RAD51D/MLH1 epistasis is specific to the processing of O6-meG lesions, the sensitivity of MEF cell lines of all the four genotypes was tested with MMS, which produces little O6-meG [3]. Rad51d−/− MEFs were sensitized to MMS compared to wild-type (Fig. 3A). Importantly, loss of MLH1 did not change the sensitivity of either Rad51d+/+Trp53−/− or Rad51d−/−Trp53−/− MEFs to MMS (Fig. 3B and C). These results support the idea that MMR-dependent sensitization of HR-deficient cells is specific for O6-meG.

Fig. 3.

Fig. 3

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Sensitivity of Rad51d-deficient MEFs to MMS is independent of MMR status. Colony survival assays of MEFs deficient in Rad51d, Mlh1 or both, treated with MMS at the indicated doses. MLH1 status does not affect the sensitivity of Rad51d-proficient or Rad51d-deficient MEFs in response to MMS [Rad51d and Mlh1-proficient (♦), Rad51d-proficient and Mlh1-deficient (○), Rad51d-deficient and Mlh1-proficient (■) and Rad51d-deficient and Mlh1-deficient (▲)]. Survival is plotted as percentage compared to untreated control. Each data point represents the mean of three independent experiments each performed in triplicates. Error bars represent the standard error of means. All MEFs are Trp53−/−.

3.3. MLH1 dependent G2/M cell cycle arrest induced by MNNG

MNNG activates a G2/M checkpoint in MMR-proficient cells during the second cell cycle [10], whereas XRCC2-deficient CHO cells arrested in the first cell cycle [18]. The authors speculated that lack of HR prevents bypass or resolution of DNA intermediates created by MutSα/MutLα recognition of O6-meG:T in the first round of replication [18]. To examine cell cycle arrest in the MEFs with a combined deficiency of HR and MMR pathways, we monitored the cell cycle distribution of Rad51d and Mlh1-deficient MEF cell lines (Supplementary Fig. S1). The percentage of cells accumulated in G2/M was quantified (Fig. 4). Rad51d+/+ Mlh1+/+ cells displayed a 1.4 fold increase in the percentage of cells in G2/M by 48 h after treatment with 1 µM MNNG (Fig. 4A). In the Rad51d−/− Mlh1+/+ cells, a 1.3 fold (P<0.05) increase was observed in the percentage of G2/M cells 24 h post MNNG treatment (Fig. 4B). At 48 and 72 h post MNNG treatment, the accumulation in G2/M persisted and was higher (2.4 fold) than that seen for the Rad51d+/+ Mlh1+/+ cells. In other words, the absence of HR hastened and exacerbated the G2/M arrest induced by MNNG. In sharp contrast, the Rad51d+/+ Mlh1−/− and Rad51d−/− Mlh1−/− cells showed no induced accumulation of G2/M cells at any time point following treatment with 1 µM MNNG (Fig. 4C and D). The results reveal that the G2/M arrest depends on functional MutSα/MutLα regardless of RAD51D status.

Fig. 4.

Fig. 4

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Cell cycle arrest of Rad51d-deficient MEFs in response to MNNG is Mlh1 dependent. Percentage of the cells in G2/M phase is shown for (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/− and (D) Rad51d−/−Mlh1−/− MEFs at indicated time points after MNNG treatment. Progression of cell cycle was followed for untreated cells and those treated with 1 µM MNNG (■ Control and □ Treatment). Percentage of the cells in the G2/M phase was calculated using MODFIT-LT software. Each data point represents the mean of three independent experiments. Error bars represent the standard error of means. Statistical significance was determined by ratio t-test (*P<0.05). All MEFs are Trp53−/−.

3.4. Absence of RAD51D increases MNNG triggered apoptosis in MMR-proficient cells

An increase in the sub-G1 population of cells was observed during the flow cytometric analysis of Rad51d−/− Mlh1+/+ MEFs (Supplementary Fig. S2), suggesting that apoptosis was occurring. Therefore, the apoptotic response of the MEFs in response to MNNG was compared to determine the consequences of HR deficiency on programmed cell death (Fig. 5). Activity of caspase-3 was measured over a time course of 96 h post treatment with 1 µM MNNG. In the Mlh1-proficient background, apoptosis was detected at 48 h in both Rad51d-proficient and deficient cells (Fig. 5A and B). In the Rad51d+/+ Mlh1+/+ cells (Fig. 5A), induction of apoptosis peaked at 48 h post MNNG treatment and declined to the background levels at the later time points because the dose of MNNG used only causes <50% death as measured by colony forming assays. In contrast, in the Rad51d−/− Mlh1+/+ cells there was a more than 2-fold induction of apoptosis at 48 h post MNNG treatment that persisted through 96 h (Fig. 5B). In the Rad51d+/+ Mlh1−/− cells, apoptosis was not induced (Fig. 5C). Rad51d−/− Mlh1−/− cells displayed a modest induction of apoptosis (P<0.05) at 48 h post MNNG treatment, but no increase at the subsequent time points (Fig. 5D). Collectively, the observations from cell cycle and apoptosis studies indicate that MNNG induces cell cycle arrest and apoptosis that depends on functional MMR. In the absence of HR, the arrest and cell death are substantially increased, which strongly suggests that HR protects against DNA damage caused by MMR recognition of O6-meG.

Fig. 5.

Fig. 5

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Activation of Caspase-3 in Rad51d and Mlh1 deficient MEFs in response to MNNG treatment. (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/−, and (D) Rad51d −/−Mlh1−/− MEFs were treated with 1 µM MNNG for the indicated times. After treatment, cells were lysed and assayed for caspase-3 activity using EnzChek Caspase-3 Assay kit (Invitrogen) (■ Control and □ Treatment). The experiment was performed in triplicates and the data represents mean±s.d. Statistical significance was determined by Student’s t-test (*P<0.05). All MEFs are Trp53−/−.

3.5. Increased accumulation of DSBs in Rad51d-deficient cells in response to MNNG treatment

Induction of γ-H2AX (phosphorylated histone 2A variant) is a marker of DSBs and has been detected following MNNG treatment [10]. γ-H2AX levels were examined by western blot in the MEFs of each representative genotype to determine the effect of HR deficiency on this marker of DSBs. Rad51d+/+ Mlh1+/+ cells showed an induction of γ-H2AX at 24 h post treatment that decreased at later time points (Fig. 6A). In contrast, the Rad51d−/− Mlh1+/+ cells displayed an induction of γ-H2AX that persisted through all time points examined, suggesting that DSBs were not being resolved in the HR-deficient cells (Fig. 6B). In the Rad51d+/+ Mlh1−/− cells, there was no induction of γ-H2AX (Fig. 6C), which was expected in MMR-deficient cells. Interestingly, in the Rad51d−/− Mlh1−/−- cells there was a smaller, delayed induction of γ-H2AX (Fig. 6D), which might be a consequence of unresolved BER intermediates caused by excision of N-methylpurines in the absence of HR (see discussion).

Fig. 6.

Fig. 6

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Kinetics of H2AX phosphorylation in MNNG treated MEFs. (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/-Mlh1+/+, (C) Rad51d+/+Mlh1−/− and (D) Rad51d−/−Mlh1−/− MEFs were treated with 1 µM MNNG for the indicated times. Total cell lysates were analyzed for phosphorylated H2AX (γ-H2AX) by western blot analysis. β-Actin was used as a control for equal protein loading. The bands visualized via western blot were subjected to band densitometry analysis using Image J software (open source Image J software available at http://rsb.info.nih.gov/ij/). The amount of specific signal for γ-H2AX was corrected for sample loading by normalization with the constitutive β-Actin signal. The value for the 0 h time point was set as 1 to calculate the Fold Induction values which were plotted as a function of time.

3.6. DNA damage signaling responses in Rad51d-deficient cells following MNNG treatment

To gain insight into how combined deficiency of HR and MMR pathways influences ATR/CHK1 damage signaling in response to MNNG, phosphorylation of CHK1 was examined. Levels of p-CHK1 (Ser345) were determined by western blot in the MEFs of each genotype following MNNG treatment over a period of 72 h and compared as a ratio to CHK1. Rad51d+/+ Mlh1+/+ cells showed an induction of p-CHK1 at 24 h post treatment, which decreased back to basal levels by 48 h (Fig. 7A). In contrast, the Rad51d−/− Mlh1+/+ cells displayed a higher basal level of p-CHK1 in the absence of treatment, and an induction at 24 h that persisted through 48 (Fig. 7B). In the Rad51d+/+ Mlh1−/− cells, there was an induction of p-CHK1 at 24 h that decreased by 48 h (Fig. 7C). Interestingly, there was a modest induction of p-CHK1 in the Rad51d−/− Mlh1−/− cells (Fig. 7D), which paralleled the induction of γ-H2AX seen in the cells deficient in both HR and MMR.

Fig. 7.

Fig. 7

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Kinetics of CHK1 phosphorylation in Rad51d and Mlh1 deficient MEFs in response to treatment with 1µM MNNG. (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/− and (D) Rad51d−/−Mlh1−/− MEFs were treated with 1 µM MNNG for the indicated times. Whole cell lysates were analyzed for total CHK1, phospho-CHK1(Ser345), by western blot analysis. β-Actin was used as a control for equal protein loading. The bands visualized via western blot were quantified by Image J software (open source Image J software available at http://rsb.info.nih.gov/ij/). The amount of p-CHK1 relative to total CHK1 was calculated as ratio.

4. Discussion

HR protects cells from killing induced by SN1-type methylating agents [16–18,29,30]. Paradoxically, the mismatch DNA repair pathway sensitizes cells to the same damage [3]. Because of the contrasting roles of HR and MMR in response to these agents, we were interested in studying the individual and collective consequences of HR and MMR deficiency for the cellular response to O6-meG. This was accomplished by intercrossing mice heterozygous for Rad51d and Mlh1 and generating the first mammalian cell lines deficient for both Rad51d and Mlh1. We observed that the Rad51d−/−Mlh1−/− embryo phenotypes were similar to Rad51d−/− embryo phenotypes in the Trp53+/+ and Trp53−/− backgrounds. These data suggest that Mlh1 deficiency does not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. This was an important first step given the established roles of HR and MMR in maintaining genome stability. One implication is that MMR and HR epistatic interactions do not occur during development in response to endogenous DNA damage.

Each of the cell lines used for these studies were Trp53-deficient, which was necessary because Rad51d−/− cells fail to proliferate [25]. Previous reports demonstrated that apoptotic signaling in response to MNNG can occur independent of p53 status [14,15]. Consistent with these findings, we observed that Mlh1-proficient Trp53-deficient cells undergo apoptotic cell death following MNNG treatment. Interestingly, all Trp53−/− MEFs showed dramatically lowered MGMT expression, which is consistent with reports that p53 regulates MGMT expression in some cell types [31–33]. In the absence of MGMT, O6-meG adducts persist [34] and are acted upon by MMR and HR.

Our studies demonstrate that MMR deficiency alleviates the hypersensitive phenotype of HR deficient cells to MNNG. The results suggest that HR resolves MMR-/O6meG-dependent DNA lesions that otherwise potently induce apoptosis. Deleting Mlh1 rescued the Rad51d-deficient hypersensitivity to MNNG by 5.2-fold, but did not fully rescue survival to that seen for Rad51d-proficient Mlh1-deficient cells. MNNG and MMS both produce 7-methylguanine and lesser amounts of 3-methyladenine, which are substrates for AAG-mediated BER [3,35]. 3-Methyladenine is a replication blocking lesion. AAG excision of 7-methylguanine, an adduct not thought to be directly mutagenic or toxic, can cause cellular sensitization to alkylating agents by creating BER strand break intermediates [36–39]. Rad51d-deficient MEFs are sensitized to MMS, which is likely due to a necessity of HR to resolve BER strand break intermediates generated by AAG excision of 7-methylguanine or to bypass a replication block caused by 3-methyladenine. Mlh1 status does not change sensitivity to MMS for Rad51 d-deficient or proficient MEFs, clearly showing that MMR is not involved in the response to damage caused by SN2-type alkylating agents in this model system. The modest induction of apoptosis by MNNG in the Rad51d Mlh1 deficient cells at 48 h is likely due to AAG-mediated excision of 7-methylguanine in the HR deficient background, whereas no apoptosis was evident in Rad51d-proficient Mlh1-deficient cells. The results are consistent with the conclusion that apoptotic signaling stemming from BER and MMR are distinct [38]. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O6-meG.

Jiricny and coworkers observed an arrest in the first cell cycle following MNNG treatment in XRCC2-deficient V-79 cells and speculated that lack of HR prevents bypass or resolution of DNA intermediates created by MutSα/MutLα recognition of O6-meG:T in the first round of replication [18]. Consistent with this, the Rad51d-deficient cells exhibited a statistically significant increased accumulation of cells in G2/M at 24 h post MNNG treatment. The increase in Rad51d-deficient cells in G2/M was dramatically higher at 48 h and 72 h post treatment. There was no G2/M accumulation in the Mlh1-deficient background, regardless of Rad51d status. These data convincingly show that the G2/M cell cycle arrest in response to MNNG is solely dependent on the presence of MMR. Moreover, the results reveal the importance of HR as a functional means of overcoming the arrest induced by MMR recognition of O6-meG in the first round of replication. These results strongly suggest that HR actively promotes bypass or resolution of DNA gap intermediates created by MutSα/MutLα recognition of O6-meG:T.

DNA damage checkpoint responses dependent on MutSα/MutLα recognition of O6-meG:T include ATR-dependent activation of CHK1 [9–11]. In the Mlh1-proficient background, the Rad51d-deficient cells displayed CHK1 phosphorylation that persisted through 48 h, whereas the phosphorylation of CHK1 peaked at 24 h in the Rad51d-proficient cells and returned to baseline by 48 h. In other words, HR deficiency exacerbates and prolongs CHK1 activation. In the Mlh1-deficient cells, there was an induction of p-CHK1 at 24 h that decreased by 48 h, demonstrating that CHK1 can be activated by MNNG treatment independent of MMR. Interestingly, a modest induction of p-CHK1 was observed in cells deficient for both Rad51d and Mlh1, yet was delayed to 48 h. The activation of CHK1 in Mlh1-deficient cells likely reveals signaling responses to BER intermediates. Induction of γ-H2AX was measured as a means of determining whether MMR recognition of O6-meG induces a cellular DSB response in the absence of HR. Rad51d and Mlh1 proficient cells showed an induction of γ-H2AX at 24 h post treatment that decreased at longer time points. In contrast, the Rad51d-deficient cells displayed a persistent induction of γ-H2AX, suggesting that DSBs remained unresolved in the HR-deficient cells. In the Rad51d-proficient Mlh1-deficient cells, there was no induction of γ-H2AX at the dose of MNNG used. Interestingly, in the cells deficient in both Rad51d and Mlh1, there was an induction of γ-H2AX, which paralleled the observations seen for p-CHK1. Therefore, future studies are required to directly address the role of BER in the Rad51d-deficient cells.

In the absence of HR, the cell cycle arrest, DSB signaling, and cell death are substantially increased, which is consistent with the futile cycling model. Specifically, the data suggest that processing of O6-meG by MMR generates DNA intermediates that are substrates for the HR machinery. The data presented in this study do not discount the participation of MMR in DNA damage signaling, but suggest that the MMR recognition and/or subsequent processing produces a DNA intermediate or secondary lesion. Consequently, there is a heightened induction of apoptosis in the Rad51d-deficient cells, suggesting that these secondary lesions are lethal if not resolved by HR. A recent study has implicated Exonuclease 1 in O6-meG-induced cell death in vivo [40], which also supports the notion that functional MMR processing of O6-meG contributes to cell death. The induction of γ-H2AX suggests that these lethal intermediates could be DSBs, although other sources of replicational stress conceivably cause the induction of γ-H2AX. However, the HR substrate created by MMR could be other DNA intermediates, such as single strand gaps [18]. Collectively, the results provide evidence that RAD51D-mediated HR is a major protective repair mechanism that paradoxically responds to a DNA damage event caused by MMR. MNNG and other SN1-type methylating agents induce SCEs at much lower doses than the doses that cause cell death [1]. It is also important to consider that the resolution of an HR event as a crossover is also the source of loss of heterozygosity, which can be procarcinogenic. Therefore, in the case of O6-meG genome damage, HR might promote cell survival at a cost of increased genome instability. Moreover, it is logical to conclude from these results that tumors with intact MMR and defective HR would be hypersensitized to clinically used methylating agents such as temozolomide. Future studies aimed at better understanding the role of HR will provide important insight into mechanisms of carcinogenesis and mechanisms of chemotherapy resulting from O6-meG adducts.

Supplementary Material

01. Supplementary Fig. S1.

Representative cell cycle distribution data of (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/−, and (D) Rad51d−/−Mlh1−/− MEFs. Progression of cell cycle was analyzed by flow cytometry for both untreated cells and those treated with 1 µM MNNG. The percentage of cells in G2/M cycle stage is indicated.

02. Supplementary Fig. S2.

The percentage of cells with sub-G1 DNA content during the flow cytometric analysis of Rad51d+/+Mlh1+/+, Rad51d−/−Mlh1+/+, ad51d+/+Mlh1−/−, and Rad51d−/− Mlh1−/− MEFs was calculated using MODFIT-LT software. The values were normalized to untreated controls and plotted as a function of time. Each data point represents the mean of three independent experiments. Error bars represent the standard error of means.

Acknowledgements

Dr. Alan Waldman is thanked for helpful discussions. This research was supported in part by a grant from the NIH to the Center for Colon Cancer Research at USC (P20 RR17698), from the NIH/NCI to MDW (R01 CA100450) and from the American Cancer Society to DLP (RSG-03-158-01-GMC).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplementary Fig. S1.

Representative cell cycle distribution data of (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/−, and (D) Rad51d−/−Mlh1−/− MEFs. Progression of cell cycle was analyzed by flow cytometry for both untreated cells and those treated with 1 µM MNNG. The percentage of cells in G2/M cycle stage is indicated.

NIHMS177831-supplement-01.ppt (753KB, ppt)
02. Supplementary Fig. S2.

The percentage of cells with sub-G1 DNA content during the flow cytometric analysis of Rad51d+/+Mlh1+/+, Rad51d−/−Mlh1+/+, ad51d+/+Mlh1−/−, and Rad51d−/− Mlh1−/− MEFs was calculated using MODFIT-LT software. The values were normalized to untreated controls and plotted as a function of time. Each data point represents the mean of three independent experiments. Error bars represent the standard error of means.

NIHMS177831-supplement-02.ppt (152.5KB, ppt)

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