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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 December 30; 291(53): 27298–27312.
Published online 2016 November 21. doi:  10.1074/jbc.M116.760561
PMCID: PMC5207156

The Major Replicative Histone Chaperone CAF-1 Suppresses the Activity of the DNA Mismatch Repair System in the Cytotoxic Response to a DNA-methylating Agent*

Abstract

The DNA mismatch repair (MMR) system corrects DNA mismatches in the genome. It is also required for the cytotoxic response of O6-methylguanine-DNA methyltransferase (MGMT)-deficient mammalian cells and yeast mgt1Δ rad52Δ cells to treatment with Sn1-type methylating agents, which produce cytotoxic O6-methylguanine (O6-mG) DNA lesions. Specifically, an activity of the MMR system causes degradation of irreparable O6-mG-T mispair-containing DNA, triggering cell death; this process forms the basis of treatments of MGMT-deficient cancers with Sn1-type methylating drugs. Recent research supports the view that degradation of irreparable O6-mG-T mispair-containing DNA by the MMR system and CAF-1-dependent packaging of the newly replicated DNA into nucleosomes are two concomitant processes that interact with each other. Here, we studied whether CAF-1 modulates the activity of the MMR system in the cytotoxic response to Sn1-type methylating agents. We found that CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to the prototypic Sn1-type methylating agent N-methyl-N′-nitro-N-nitrosoguanidine. We also report evidence that in human MGMT-deficient cell-free extracts, CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system. Taken together, these findings suggest that CAF-1-dependent incorporation of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, thereby defending the cell against killing by the Sn1-type methylating agent.

Keywords: cancer, DNA mismatch repair, DNA replication, genomic instability, histone chaperone, mutL homolog 1 (MLH1)

Introduction

The DNA mismatch repair (MMR)2 system has been conserved from bacteria to humans as a consequence of its importance for the maintenance of genome stability (1,3). The MMR system has several activities that are involved in genome metabolism (1, 3,10). The correction of DNA mismatches is the best understood activity of the MMR system. Eukaryotic MMR leads to the correction of errors caused by the replication fork DNA polymerases α, δ, and ϵ, and it also changes the outcome of homologous recombination (11,17). A considerable number of proteins have been suggested to contribute to or regulate eukaryotic MMR (6, 18,20). Strong evidence indicates the primary mismatch recognition factor MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in budding yeast) endonuclease, the replicative clamp proliferating cell nuclear antigen (PCNA), the clamp loader replication factor C (RFC), the exonuclease EXO1, the secondary mismatch recognition factor MutSβ (MSH2-MSH3 heterodimer), and the replicative DNA polymerase δ (Pol δ) play major roles in eukaryotic MMR (21,39).

Several eukaryotic MMR reactions have been described (10, 36, 40,43). One of these reactions removes mismatches on both the 3′- and 5′-nicked DNA molecules in an excision-dependent manner and is probably necessary for the majority of MMR events in wild-type cells (36). This MMR reaction depends on the activities of MutSα, MutLα, PCNA, RFC, EXO1, RPA, and Pol δ (32, 33, 35,37, 41) and is initiated by recognition of the mismatch by MutSα (21, 22). After recognizing the mismatch, MutSα cooperates with RFC-loaded PCNA to activate MutLα endonuclease (37, 41, 44). The activated MutLα endonuclease incises the discontinuous daughter strand. An incision that is generated by MutLα endonuclease 5′ to the mismatch is used by EXO1 to enter the DNA and excise the mismatch in a 5′ → 3′-directed reaction modulated by MutSα and RPA (33, 35, 37, 45). A gap generated by EXO1 action is filled in by Pol δ holoenzyme. Although Pol ϵ holoenzyme can also perform gap filling in a reconstituted excision-dependent MMR reaction, the specific activity of this enzyme is much lower than that of Pol δ holoenzyme (43). The mutator phenotype of an exo1Δ mutant is weaker compared with the mutator phenotype of an msh2Δ or mlh1Δ mutant, indicating that the MMR system can remove DNA polymerase errors in EXO1-deficient cells (14, 28, 34). In agreement with this idea, an excision-independent MMR reaction that involves MutSα, MutLα, PCNA, RFC, RPA, and Pol δ has been described (41). Like the excision-dependent MMR reaction, the excision-independent MMR reaction requires the MutLα endonuclease activity for incision of the discontinuous daughter strand. After MutLα endonuclease incises the discontinuous daughter strand, the Pol δ holoenzyme uses a MutLα-generated 3′ end that is 5′ to the mismatch to perform strand displacement DNA synthesis that removes the mismatch. The role of the Pol δ holoenzyme in the excision-independent MMR reaction may be unique because the replacement of the Pol δ holoenzyme with the Pol ϵ holoenzyme abolishes the excision-independent MMR reaction (43).

One of the activities of the MMR system is required for the cytotoxic response of MGMT (O6-methyl guanine methyl transferase)-deficient mammalian cells and yeast mgt1Δ rad52Δ cells to Sn1-type methylating agents (46, 47). This activity of the MMR system depends on MutSα and MutLα and is necessary for several therapies against MGMT-deficient cancers (3). The treatment involves dacarbazine, procarbazine, or temozolomide, each of which is an Sn1-type methylating drug that triggers death of MGMT-deficient cancer cells by activating the MMR system. O6-Methylguanine (O6-mG) is the cytotoxic product of treatment of the cell with the Sn1-type methylating agent (48). Normally, O6-mG lesions are removed by MGMT that protects the cell from killing by the Sn1-type methylating agent (49). However, a significant number of cancers are deficient in MGMT due to methylation of the MGMT promoter (50). MGMT-deficient cancer cells treated with the Sn1-type methylating agent accumulate O6-mG-T mispairs that are recognized by MutSα (51). Upon recognition of an O6-mG-T mispair, MutSα initiates its repair. If the O6-mG is on the discontinuous strand, it gets repaired (52). In contrast, if the O6-mG is on the continuous strand, it triggers futile cycles of MMR (52). The futile cycles of MMR lead to the formation of persistent strand breaks (53), which are converted in the next S phase into double strand breaks that cause cell cycle arrest followed by cell death (54, 55). Consistent with this, double strand break repair defects sensitize eukaryotic cells to the killing effects of Sn1-type methylating agents (47, 56).

The heterotrimeric CAF-1 is the major histone chaperone for the assembly of nucleosomes onto the newly replicated DNA (57,61). CAF-1 loads histone (H3-H4)2 tetramers onto DNA, producing tetrasomes (62, 63). Each tetrasome is then converted into a nucleosome by the addition of two histone H2A-H2B dimers (57, 64). CAF-1 interacts physically with PCNA, and this interaction is necessary for the action of CAF-1 on the newly replicated DNA (65, 66). Recent research has indicated that postreplicative MMR coincides with CAF-1-dependent nucleosome assembly and that the two processes interact with each other (43, 63, 67). Furthermore, recent findings are consistent with the idea that the eukaryotic MMR system degrades irreparable O6-mG-T mispair-containing DNA when it is being packaged into nucleosomes by the CAF-1-dependent mechanism (53, 63, 67). We show here that CAF-1 suppresses the activity of the MMR system in the cytotoxic response to Sn1-type methylating agents.

Results

CAF-1 Suppressed the Activity of the MMR System in the Cytotoxic Response of Yeast mgt1Δ rad52Δ Cells to MNNG

The cytotoxic response to Sn1-type methylating agents occurs in the chromatin environment (46, 52, 53, 68). However, it has remained unknown whether the chromatin environment affects the cytotoxic response to Sn1-type methylating agents. It has also been unknown whether histone chaperones, proteins that are involved in the control of chromatin environment, influence the cytotoxic response to Sn1-type methylating agents. We wanted to study whether the major replicative histone chaperone CAF-1 impacts the cytotoxic response to an Sn1-type methylating agent. Previous research showed that MGMT-deficient mammalian cells and Saccharomyces cerevisiae mgt1Δ rad52Δ that lack Mgt (the yeast ortholog of MGMT (69)) and the recombination mediator Rad52 (70) are efficiently killed by the prototypic Sn1-type methylating agent MNNG in a manner that involves the MMR system (46, 47, 53). Thus, we utilized the yeast mgt1Δ rad52Δ cells to determine whether CAF-1 impacts the cytotoxic response to MNNG. In budding yeast, CAC1 encodes the largest subunit of CAF-1 (60). Accordingly, we investigated whether loss of the CAC1 gene increased the sensitivity of the yeast mgt1Δ rad52Δ cells to killing by MNNG. The use of an MNNG cytotoxicity assay permitted us to establish that the cac1Δ mgt1Δ rad52Δ cells were more sensitive to treatment with MNNG than the mgt1Δ rad52Δ cells (Fig. 1A). A more detailed analysis revealed that ~1.7% of the mgt1Δ rad52Δ cells and only ~0.2% of the cac1Δ mgt1Δ rad52Δ cells survived the treatment with MNNG (Fig. 1C). Thus, the surviving fraction of the MNNG-treated cac1Δ mgt1Δ rad52Δ cells was ~9 times smaller than that of the MNNG-treated mgt1Δ rad52Δ cells. We then conducted experiments to determine whether MNNG killed the cac1Δ mgt1Δ rad52Δ cells via an MMR system-dependent mechanism (Fig. 1C). The results showed that the deletion of the MMR system gene MLH1 rescued the sensitivity of the cac1Δ mgt1Δ rad52Δ cells to the cytotoxic effect of MNNG. The experiments also demonstrated that the mlh1Δ cac1Δ mgt1Δ rad52Δ cells were as resistant to the MNNG treatment as the mlh1Δ mgt1Δ rad52Δ cells. Based on these findings, we concluded that loss of CAC1 sensitizes the yeast mgt1Δ rad52Δ cells to MMR system-dependent killing by MNNG.

FIGURE 1.
CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to MNNG. Cytotoxicity assays were carried out as detailed under “Experimental Procedures.” A and B, cytotoxic responses ...

The cytotoxicity of Sn1-type methylators is mediated by replication- and MMR system-dependent double strand breaks that these agents form. A number of other DNA-damaging agents generate strand breaks that kill cells. Among them are camptothecin, hydroxyurea, and bleomycin. Camptothecin induces replication-dependent double strand breaks by stabilizing topoisomerase I-DNA covalent complexes (71), hydroxyurea causes double strand breaks by depleting the dNTP pools (72), and bleomycin creates double strand and single strand breaks by attacking DNA (73). Unlike the Sn1-type methylating agent, camptothecin, hydroxyurea, and bleomycin kill cells via MMR system-independent mechanisms. Nevertheless, there is a significant similarity between one of these drugs, camptothecin, and MNNG in that double strand breaks caused by these two agents are formed during DNA replication. The results of our genetic experiments (Fig. 1, A and C) raised the possibility that loss of CAC1 sensitized the yeast mgt1Δ rad52Δ cells to the cytotoxic effects of DNA-damaging agents that generate DNA breaks. To address this possibility, we studied the effect of CAC1 absence on the sensitivity of the mgt1Δ rad52Δ cells to camptothecin, hydroxyurea, and bleomycin (Fig. 1, D and E). An analysis of the data showed that the cac1Δ mgt1Δ rad52Δ cells and the mgt1Δ rad52Δ cells had the same sensitivities to bleomycin, camptothecin, and hydroxyurea. Thus, CAC1 absence did not affect the sensitivity of the yeast mgt1Δ rad52Δ cells to bleomycin, camptothecin, and hydroxyurea, drugs that kill cells via MMR system-independent mechanisms. Because MNNG kills the yeast mgt1Δ rad52Δ cells by activating an MMR system-dependent mechanism and bleomycin, camptothecin, and hydroxyurea kill the cells via other mechanisms, the results of our genetic experiments (Fig. 1, A and C–E) indicated that Cac1 increases the survival of the MNNG-treated mgt1Δ rad52Δ cells by being involved in a process that suppresses the cytotoxic activity of the MMR system.

CAC2 codes for the second subunit of yeast CAF-1 (60). We studied whether deletion of CAC2 sensitized the mgt1Δ rad52Δ cells to MNNG (Fig. 1B). The data showed that the cac2Δ mgt1Δ rad52Δ cells were more sensitive to MNNG than the mgt1Δ rad52Δ cells (Fig. 1, B and C). A comparison of the sensitivities of the cac2Δ msh2Δ mgt1Δ rad52Δ, msh2Δ mgt1Δ rad52Δ, cac2Δ mgt1Δ rad52Δ, and mgt1Δ rad52Δ cells (Fig. 1C) revealed that MNNG killed the cac2Δ mgt1Δ rad52Δ cells via an MMR system-dependent mechanism. As expected, the cac2Δ mgt1Δ rad52Δ cells were as sensitive to camptothecin and hydroxyurea as the mgt1Δ rad52Δ cells (Fig. 1F). Thus, the results of these and previous experiments (Fig. 1, A–F) demonstrated that CAF-1 increases the survival of MNNG-treated mgt1Δ rad52Δ cells by suppressing the cytotoxic activity of the MMR system.

Loss of HIR2, RTT106, or HHT2-HHF2 Did Not Change the Sensitivity of Yeast mgt1Δ rad52Δ Cells to MNNG

Histone H3-H4 chaperone HIR (Hir1-Hir2-Hir3-Hpc2 complex) plays a major role in replication-independent nucleosome assembly (74), and histone H3-H4 chaperone Rtt106 participates in replication-coupled nucleosome assembly (75). We analyzed whether the absence of HIR2 or RTT106 had an effect on the sensitivity of the mgt1Δ rad52Δ cells to MNNG. The experiments showed that lack of HIR2 or RTT106 did not change the sensitivity of the mgt1Δ rad52Δ cells to MNNG (Fig. 1G).

The molecular activity of CAF-1 is to load histone H3-H4 tetramers onto newly replicated DNA (59, 62, 64). We wanted to explore whether decreasing histone H3-H4 gene dosage affected the survival of MNNG-treated mgt1Δ rad52Δ cells. The yeast genome contains two histone H3-H4 gene loci, HHT1-HHF1 and HHT2-HHF2. Compared with HHT1-HHF1, HHT2-HHF2 produces about 5–7 times more mRNA (76). Either locus is sufficient to maintain the existence of the yeast cell. We measured the sensitivity of hht2-hhf2Δ mgt1Δ rad52Δ cells to MNNG and found it to be no different from that of the mgt1Δ rad52Δ cells (Fig. 1G).

CAF-1-dependent Packaging of Irreparable O6-mG-T Mispair-containing DNA into Nucleosomes Suppressed Its Degradation by the MMR System

MMR system-dependent degradation of irreparable O6-mG-T mispair-containing DNA is involved in the cytotoxic response to the Sn1-type methylating drug (52, 53, 68). Our genetic experiments indicated that CAF-1 activity suppresses degradation of irreparable O6-mG-T mispair-containing DNA by the MMR system (Fig. 1). To find evidence that CAF-1-dependent incorporation of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, we performed biochemical experiments that are summarized in Figs. 227; DNA substrates that we utilized in these experiments were 3′-nicked O6-mG-T (3O6-mG-T), 3′-nicked G-T (3G-T), and 3′-nicked A-T (3A-T) DNAs. The substrates were made using a plasmid, pAH1A, as a starting material and differed from each other by 1–2 bases (52). The 3′-nicked O6-mG-T DNA contained a single O6-mG-T mispair, the 3′-nicked G-T DNA carried a single G-T mispair, and the 3′-nicked A-T DNA lacked a mispair. The 3′-nicked O6-mG-T DNA was irreparable by the MMR system because the O6-mG was on the continuous strand. In these biochemical experiments, we used a cytosolic extract prepared from human embryonic kidney cell line 293T that lacked MutLα and MGMT (77, 78) and had a reduced level of CAF-1 (Fig. 2A) (58). Although the 293T cytosolic extract lacks the MMR system due to the absence of MutLα (77), supplementation of the extract with purified MutLα reconstitutes the MMR system (78). In agreement with previous studies (37, 52, 63, 77,79), our control experiments showed that 1) the reconstituted MMR system failed to repair an O6-mG-T mispair on a nicked DNA (3′-nicked O6-mG-T DNA) but repaired a G-T mispair on a similar nicked DNA (the 3′-nicked G-T DNA) (Fig. 3), 2) supplementation of the 293T cytosolic extract with purified MutLα-E705K endonuclease mutant (37) did not lead to reconstitution of the MMR system (Fig. 3), 3) the omission of exogenous dNTPs and the addition of the DNA polymerase inhibitor aphidicolin led to a nearly complete inhibition of the mismatch correction activity of the reconstituted MMR system (Fig. 3), 4) the 293T cytosolic extract had a weak nucleosome assembly activity (Fig. 2B) due to the reduced level of CAF-1 (Fig. 2A).

FIGURE 2.
Nucleosome assembly reactions reconstituted with an extract and purified CAF-1. The experiments were conducted and analyzed as described under “Experimental Procedures.” A, analysis of CAF-1 in 293T cytosolic extract (293T CE) and 293T ...
FIGURE 3.
MMR system reconstituted with an extract and purified MutLα does not correct a mispair that contains O6-mG on the continuous strand. The experiments were carried out and analyzed as described under “Experimental Procedures.” Each ...
FIGURE 4.
MMR system reconstituted with an extract and purified MutLα degrades the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA. The experiments were carried out as described in the legend to Fig. 3. The recovered DNAs were digested ...
FIGURE 5.
CAF-1-dependent suppression of persistent MutLα endonuclease-dependent degradation of an irreparable O6-mG-T mispair-containing DNA in an extract system. The experiments were carried out and analyzed as described under “Experimental Procedures.” ...
FIGURE 6.
CAF-1 suppresses persistent degradation of an irreparable O6-mG-T mispair-containing DNA in an extract system in a concentration-dependent manner. The experiments were carried out and analyzed as detailed under “Experimental Procedures.” ...
FIGURE 7.
CAF-1-dependent suppression of the formation of a gap in an irreparable O6-mG-T mispair-containing DNA in an extract system. The experiments were performed and analyzed as described under “Experimental Procedures.” Each reaction mixture ...

We next utilized Southern hybridization to detect MMR system-dependent degradation of the irreparable O6-mG-T mispair-containing DNA that was reconstituted with the 293T cytosolic extract in the absence or presence of the purified CAF-1 (Figs. 447). The initial experiments in this series analyzed degradation products that were separated on denaturing agarose gels (Figs. 446). The data showed that the reconstituted MMR system degraded the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA (the 3′-nicked O6-mG-T DNA) leading to the formation of a ~130-nt product (Figs. 4A and and55A, lane 10). Importantly, the ~130-nt product was not observed in the reaction mixture that contained endonuclease-deficient MutLα-E705K instead of MutLα (Figs. 4A and and55A, lane 13) demonstrating that the endonuclease activity of MutLα is required for the degradation of the 3′-nicked O6-mG-T DNA. Additional experiments revealed that the ~130-nt product or a similar product was not formed from the 3′-nicked A-T DNA in the reaction mixture that contained the reconstituted MMR system (Figs. 4A and and55A, lane 17). The results of a time course analysis demonstrated that the ~130-nt product of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA was observed in the reaction mixture that was incubated for 10–120 min (Fig. 5B). This observation indicated that the reconstituted MMR system caused persistent degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA. In contrast, the products of degradation of the discontinuous strand of the 3′-nicked G-T DNA that were present in the reaction mixture incubated for 10 min (Fig. 4A, lane 3) were not detected in the same reaction mixture that was incubated for 60 min (Fig. 5A, lane 3). This finding is consistent with the view that the product of degradation of the discontinuous strand of the 3′-nicked G-T DNA is an intermediate of the MMR reaction (37).

The ~130-nt product was identified by Southern hybridization with a 32P-labeled probe that was complementary to a discontinuous strand sequence located 2 nt downstream from the mismatched T (Figs. 4A and and55A). However, Southern hybridization with a 32P-labeled probe that was complementary to a discontinuous strand sequence located 2 nt upstream from the mismatched T did not detect the ~130-nt product (Fig. 5C). This finding indicated that the 5′ end of the ~130-nt product was located at or near the mismatch. To determine whether a different part of the 3′-nicked O6-mG-T DNA contained a MutLα endonuclease-dependent strand break, we carried out Southern hybridizations with two other 32P-labeled probes (Fig. 5, D and E). One of the probes was complementary to a discontinuous strand sequence that was downstream from the preexisting strand break (Fig. 5D) and the other to a continuous strand sequence (Fig. 5E). These two Southern hybridizations did not identify any additional MutLα endonuclease-dependent strand break in the 3′-nicked O6-mG-T DNA.

The addition of purified CAF-1 to the 293T extract- and MutLα-containing reaction mixture led to packaging of the 3′-nicked O6-mG-T DNA into nucleosomes (Fig. 2B, lane 9). The presence of purified CAF-1 in the 293T extract- and MutLα-containing reaction mixture also caused a significant decrease in the yield of the ~130-nt product of degradation of the irreparable O6-mG-containing DNA (Fig. 5, A and B). The effect of CAF-1 on the yield of the ~130-nt product was especially pronounced in the reaction mixture in which the concentration of the 3′-nicked O6-mG-T DNA was decreased 4 times (Fig. 5F). In this case, the addition of purified CAF-1 reduced the yield of the ~130-nt degradation product 5-fold. In the above experiments, we included 2.4 pmol of purified CAF-1 in the 293T extract- and MutLα-containing reaction mixtures. Further experiments showed that the addition of 0.3 pmol of purified CAF-1 was sufficient to cause a significant decrease in the yield of the ~130-nt product (Fig. 6, A and B). The simplest interpretation of these results is that CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes reduces its degradation by the MMR system.

We also utilized Southern hybridization to analyze the degradation products that were separated on native agarose gels (Fig. 7). The data revealed that in the 293T extract- and MutLα-containing reaction mixture, a significant fraction of the 3′-nicked O6-mG-T DNA was converted into a gapped product (Fig. 7). A gap was formed in the discontinuous but not continuous strand of the 3′-nicked O6-mG-T DNA (Fig. 7, B and C, lane 7). The same or a similar gapped product was largely absent in the 293T extract- and MutLα-E705K-containing mixture (Fig. 7B, lane 10). Although some of the 3′-nicked G-T DNA products in the 293T extract- and MutLα-containing reaction mixture were also gapped (Fig. 7B, lane 2), their yield was ~2.5 times lower than that of the gapped product of the 3′-nicked O6-mG-T DNA (Fig. 7D). As expected, 3′-nicked A-T DNA products that were formed in the 293T extract- and MutLα-containing reaction mixture lacked gaps (Fig. 7, B (lane 12) and C). Supplementation of the 293T extract- and MutLα-containing reaction mixture with 0.6 or 2.4 pmol of purified CAF-1 caused a significant reduction in the yield of the gapped product of the 3′-nicked O6-mG-T DNA (Fig. 7, B–E). This finding supports the view that CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system.

Degradation of an Irreparable O6-mG-T Mispair-containing DNA by the Activated MutLα Endonuclease in a Defined System

Our biochemical experiments (Figs. 447) have implicated MutLα endonuclease activity in MMR system-dependent degradation of the discontinuous strand of irreparable O6-mG-T mispair-containing DNA. However, these experiments did not provide a clear view of how MutLα endonuclease activity is involved in this process. To better understand the involvement of MutLα endonuclease activity in MMR system-dependent degradation of the discontinuous strand of irreparable O6-mG-T mispair-containing DNA, we carried out additional experiments summarized in Fig. 8. In these experiments, we studied degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA in a defined system (37). Purified proteins that were included in the defined system were MutLα endonuclease, the mismatch recognition factor MutSα, the PCNA clamp, the RFC clamp loader, and the single-stranded DNA-binding protein RPA. It can be seen that the discontinuous strand of the 3′-nicked O6-mG-T DNA was degraded in the defined system in a MutLα endonuclease concentration-dependent manner (Fig. 8, A (lanes 13–17) and B). The level of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA was 5–6 times higher than the degradation level of the discontinuous strand of the control 3′-nicked A-T DNA (Fig. 8B). No degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA was observed in the defined system in which the endonuclease-deficient MutLα-E705K substituted for MutLα (Fig. 8, A (lanes 6, 12, and 18) and C). This information indicated that MutLα provided the endonuclease activity that degraded the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA in the presence of MutSα, PCNA, RFC, and RPA. Importantly, the ~130-nt fragment that was detected in the cell extract system (Fig. 5A, lane 10) was not a preferred product of the endonuclease reaction in the defined system (Fig. 8A, lanes 15–17). This is an indication that the defined system lacks one or more factors that are involved in the formation of the ~130-nt fragment in the extract system. While degrading the discontinuous strand, MutLα endonuclease did not incise the continuous strand of the 3′-nicked O6-mG-T DNA (Fig. 8E, lanes 13–17). An inspection of the data in Fig. 8A showed that MutLα endonuclease incised the discontinuous strand of the 3′-nicked O6-mG-T DNA at random sites, displaying no significant site or sequence specificity. These findings suggested that MutLα endonuclease contributes to MMR system-dependent degradation of irreparable O6-mG-T mispair-containing DNA by introducing strand breaks at random sites on the discontinuous strand. In addition, these findings suggested that processing of the MutLα endonuclease-incised discontinuous strand of the 3′-nicked O6-mG-T DNA in the extract system led to the formation of the ~130-nt product (Fig. 4A, lane 10). In agreement with this idea, the pattern of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA in the extract system that was supplemented with the DNA polymerase inhibitor aphidicolin (Fig. 4A, lane 14) is similar to the pattern of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA in the defined system (Fig. 8A, lanes 13–17).

FIGURE 8.
MutLα endonuclease-dependent degradation of an irreparable O6-mG-T mispair-containing DNA in a defined system. The experiments were performed and analyzed as described under “Experimental Procedures.” Each reaction mixture contained ...

Discussion

Mammalian MGMT-deficient and yeast mgt1Δ rad52Δ cells are efficiently killed by low doses of Sn1-type methylating agents (46, 47, 78, 80,82). Several anticancer therapies exploit the marked sensitivity of MGMT-deficient cells to Sn1-type methylating agents. The marked sensitivity of mammalian MGMT-deficient and yeast mgt1Δ rad52Δ cells to Sn1-type methylating agents is a result of the MMR system-dependent cytotoxic response (46, 47, 78, 80,82). Previous research showed that the cytotoxic response to the Sn1-type methylating drug involves MMR system-dependent degradation of irreparable O6-mG-containing DNA that leads to the formation of lethal double strand breaks (47, 53, 56). The MMR system starts to degrade irreparable O6-mG-containing nascent DNA behind the replication fork (53). At the same time, this DNA is incorporated into nucleosomes in the CAF-1-orchestrated process (57,61). It was previously unknown whether concomitant CAF-1-dependent nucleosome assembly affects degradation of irreparable O6-mG-containing DNA by the MMR system. We have found that CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to MNNG (Fig. 1). We have also found that in an MGMT-deficient extract system, CAF-1-dependent incorporation of an irreparable O6-mG-T mispair-containing DNA into nucleosomes (Fig. 2B) correlates with a substantial decrease in degradation of this DNA by a MutLα endonuclease-dependent mechanism (Figs. 557). These findings imply that CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, therefore defending the cell against killing by the Sn1-type methylating drug. Consistent with this, we have established that loss of CAF-1 does not affect the sensitivity of yeast mgt1Δ rad52Δ cells to bleomycin, camptothecin, and hydroxyurea, DNA-damaging drugs that kill cells in an MMR system-independent manner (Fig. 1). It is known that 1–2 double strand breaks are sufficient to kill the yeast rad52 cell (83). Therefore, the fact that the MNNG treatment kills the cac1Δ mgt1Δ rad52Δ and cac2Δ mgt1Δ rad52Δ cells more efficiently than the mgt1Δ rad52Δ cells indicates that CAF-1 loss increases the fraction of MNNG-treated cells that experience at least 1–2 double strand breaks.

In addition to CAF-1, S. cerevisiae cells contain several other histone chaperones, including HIR and Rtt106 (75). Our results indicated that loss of HIR2 or RTT106 does not increase the sensitivity of mgt1Δ rad52Δ cells to MNNG (Fig. 1G). Thus, these results imply that neither of these histone chaperones has a non-redundant function that provides a protection for mgt1Δ rad52Δ cells from the cytotoxic activity of the MMR system. We determined that decreasing histone H3-H4 gene dosage by deletion of the HHT2-HHF2 locus in the mgt1Δ rad52Δ cells does not change their sensitivity to MNNG (Fig. 1G). A previous report described that hht2-hhf2Δ does not affect the level of the chromatin H3-H4 histones but decreases the level of the soluble H3-H4 histones 2-fold (84). Thus, it appears that a small decrease in the level of the soluble histones H3-H4 does not affect the cytotoxic activity of the MMR system.

An earlier study has implicated MutLα endonuclease activity in the cytotoxic response to the Sn1-type methylating drug in the yeast and mammalian cells (68). We have now shown that activated MutLα endonuclease degrades the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA in an extract system and in a purified system (Figs. 448). This information implies that MutLα endonuclease-dependent degradation of the discontinuous strand of irreparable O6-mG-T mispair-containing nuclear DNA is involved in the cytotoxic response to the Sn1-type methylating drug. MutLα endonuclease degrades the 3′-nicked O6-mG-T and G-T DNA substrates in the presence of MutSα, PCNA, RFC, and RPA in a very similar way (Fig. 8A, lanes 3–5 and lanes 15–17), suggesting that the same mechanism activates MutLα endonuclease in MMR and in the cytotoxic response to the Sn1-type methylating drug. The mechanism of activation of MutLα endonuclease in MMR requires the presence of MutSα and a mismatch (37, 39, 42) (Fig. 8). The O6-mG-T mispair is one of many mispairs recognized by MutSα (21). A crystallographic study determined that MutSα has the same structure in the MutSα-G-T DNA and MutSα-O6-mG-T DNA crystals (85). This information and the results of our analysis of the defined reactions (Fig. 8) suggest that adoption of the same structure by MutSα on the G-T mispair and on the irreparable O6-mG-T mispair permits the protein to convey the same activating signal to MutLα endonuclease during MMR and the cytotoxic response to the Sn1-type methylating drug. Although the major product of persistent MutLα endonuclease-dependent degradation of the 3′-nicked O6-mG-T DNA in the extract system has a size of ~130 nt (Figs. 4A and and55A (lane 10) and 6A (lane 8)), the products of MutLα endonuclease-dependent degradation of the 3′-nicked O6-mG-T DNA in the extract system that was supplemented with aphidicolin have sizes in the range of 100–2,000 nt (Fig. 4, A and B, lane 14). Because aphidicolin is an inhibitor of the biosynthetic activities of Pol δ and Pol ϵ, these findings suggest that DNA synthesis and ligation that occur on the MutLα-degraded DNA in the extract system in the absence of aphidicolin are necessary to remove the majority of the 100–2,000-nt products.

It is important to note that a previous work described that MutLα-dependent processing of the 3′-nicked O6-mG-T DNA in the HCT116BBR nuclear extract-containing system leads to the formation of a ~130-nt product (52) that does not appear to be different from the one that we have detected in our extract system (Figs. 447). This observation implies that reactions that occur in different extract systems generate the same product of degradation of the 3′-nicked O6-mG-T DNA. We have observed that MutLα endonuclease-dependent degradation of the 3′-nicked O6-mG-T DNA in our extract system leads to the formation of a gapped product (Fig. 7). The gap was generated in the discontinuous strand in the presence of dNTPs and was located downstream from the mispaired T. Thus, the MMR system-dependent processing of the 3′-nicked O6-mG-T DNA in the cell extract generates two kinds of DNA products; one of them carries the gap (Fig. 7), and the other contains the ~130-nt fragment (Figs. 5A and and66A). It is likely that the gap is formed when the excision step is not blocked by the O6-mG, whereas the ~130-nt fragment is formed when the excision step is blocked by the lesion. The presence of a gap in the irreparable DNA is in good agreement with a previous study that documented that gaps are formed behind replication forks in response to treatment of both mammalian MGMT-deficient cells and yeast mgt1Δ rad52Δ cells with MNNG (53). The fact that the gap was generated downstream from the mismatched T (Fig. 7) suggests that O6-mG is a stronger block for the DNA polymerization reaction than for the excision reaction.

The Sn1-type methylating drug temozolomide is used for treatment of glioblastoma patients. However, recurrent glioblastomas often arise during post-treatment period (86). This indicates that an approach that increases the sensitivity of MGMT-deficient tumors to treatment with the Sn1-type methylating drug might suppress recurrent cancers. More research is needed to determine whether defective replication-coupled nucleosome assembly increases the sensitivity of MGMT-deficient cancer cells to treatment with the Sn1-type methylating drug.

Experimental Procedures

Yeast Strains

S. cerevisiae strains that were used in this study were derivatives of the wild-type haploid strain E134 (MATα ade5-1 lys2::InsE-A14 trp1-289 his7-2 leu2-3,112 ura3-52) (13). The lithium acetate/PEG4000/DMSO transformation method and PCR-amplified disruption cassettes were used to generate the strains.

MNNG and Bleomycin Cytotoxicity Assays

Liquid YPDAU medium (1% yeast extract, 2% Bacto-peptone, 2% dextrose, 60 mg/liter adenine, 60 mg/liter uracil), YPDAU plates, 1 mm stock solutions of MNNG (Wako Chemicals USA) in DMSO, a 2 mg/ml stock solution of bleomycin (Santa Cruz Biotechnology) in DMSO, and DMSO were used in the assays. Yeast cultures were grown to saturation in liquid YPDAU for ~20 h at 30 °C. The saturated cultures were diluted 10-fold in fresh medium and grown for 4 h at 30 °C. The cultures were then diluted with fresh medium to A600 = 1.3, and aliquots of the diluted cultures were treated with 1 μm MNNG, 0.1% DMSO (vehicle control in the MNNG toxicity assay), 30 μg/ml bleomycin, and 1.5% DMSO (vehicle control in the bleomycin cytotoxicity assay) for 2 h at 30 °C. After treatment, the cultures were diluted, and appropriate dilutions of the cultures were spread on YPDAU plates. The plates were incubated for 3–4 days at 30 °C, and colonies were counted. A somewhat different MNNG cytotoxicity assay was also used in this study. In this assay, 10-fold serial dilutions of the treated cultures were made and spotted on YPDAU plates. The plates were incubated for 2 days at 30 °C and photographed.

Camptothecin and Hydroxyurea Cytotoxicity Assays

Yeast cultures were grown to saturation as described above and diluted to A600 = 1.4 with sterile water. 10-fold serial dilutions of the cultures were prepared and spotted on YPDAU plates, YPDAU plates containing 0.5 μg/ml camptothecin (Enzo Life Science), and YPDAU plates containing 10 mm hydroxyurea (US Biological). After incubation for 2 days at 30 °C, the plates were photographed.

Cell Extract and Recombinant Proteins

293T cells were grown as an attached culture in DMEM/high glucose medium that was supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.29 μg/ml l-glutamine. Cytosolic and nuclear extracts from proliferating 293T cells were prepared according to a described procedure (63). Recombinant human CAF-1, MutSα, MutLα, MutLα-E705K, PCNA, RFC, and RPA were isolated in nearly homogeneous forms as described previously (37, 63).

Western Blotting

Samples of the purified CAF-1, 293T cytosolic extract (15 μg), and 293T nuclear extract (15 μg) were separated on a denaturing SDS gel and transferred onto a PVDF membrane. After the protein transfer step, the membrane was incubated with α-CAF-1 p150 antibodies (catalog no. sc-10772, lot E2004, rabbit polyclonal IgG, Santa Cruz Biotechnology, Inc.) and then with ECL HRP-conjugated secondary antibodies (catalog no. NA934V, lot 389592, donkey antibody, GE Healthcare). Immune complexes were visualized utilizing ECL2 Western blotting substrate (Thermo Fisher Scientific) and a CCD camera. Amounts of CAF-1 in the samples of the 293T cytosolic and nuclear extracts were measured by quantification of the data with the ImageJ software.

DNA Substrates, Oligonucleotides, and 32P-Labeled Hybridization Probes

3′-Nicked O6-mG-T, 3′-nicked G-T, and 3′-nicked A-T DNAs were essentially prepared as described previously (52), except that after DNA ligation, unligated material was degraded by exonuclease III (New England Biolabs), and the remaining DNA was purified by chromatography on BND-cellulose (Sigma). The O6-mG-containing oligonucleotide that was used to prepare the 3′-nicked O6-mG-T DNA was synthesized by Midland Certified Reagent Co. All other oligonucleotides used in this study were synthesized by IDT. To prepare a 32P-labeled hybridization probe, the oligonucleotide was labeled at the 5′ end with 32P by T4 polynucleotide kinase in the presence of [γ-32P]ATP. The sequences of oligonucleotides used to construct the DNA substrates and prepare 32P-labeled hybridization probes are shown in Table 1.

TABLE 1
Oligonucleotides that were used to prepare the DNA substrates and 32P-labeled hybridization probes

Nucleosome Assembly Reactions in 293T Cytosolic Extract-containing Mixtures

The nucleosome assembly reactions were carried out at 37 °C in 40-μl mixtures that contained 20 mm HEPES-NaOH (pH 7.4), 100 mm KCl, 8 mm MgCl2, 2 mm DTT, 0.2 mg/ml BSA, 0.1 mm each of the four dNTPs, 3 mm ATP, 20 mm creatine phosphate, 0.02 mg/ml creatine phosphokinase, 1% glycerol (v/v), 75 μg of 293T cytosolic extract, purified CAF-1 (0 or 1.2 pmol), MutLα (0 or 1.6 pmol), and 81 fmol (0.1 μg) of a 3′-nicked DNA (the 3′-nicked O6-mG-T DNA, the 3′-nicked G-T DNA, or the 3′-nicked A-T DNA). After 60 min of incubation, a 35-μl fraction of each reaction mixture was mixed with a 5-μl mixture containing 20 mm HEPES-NaOH (pH 7.4), 64 mm CaCl2, 8 units/μl micrococcal nuclease, and 0.02 mg/ml RNase A and incubated for 20 min at 21–23 °C. Each reaction mixture was then supplemented with a 10-μl solution containing 0.5% SDS, 150 mm EDTA, 40% glycerol, and 2 mg/ml Proteinase K, followed by incubation of the mixture for 20 min at 50 °C. Proteinase K in the mixtures was inactivated by the addition of PMSF to a final concentration of 0.8 mm. The nucleosome assembly products were separated on native 1.7% agarose gels, transferred onto nitrocellulose membranes, and hybridized with 32P-labeled probe c154. Indirectly labeled DNA species were visualized with a Typhoon biomolecular imager (GE Healthcare) and quantified with ImageQuant software.

Mismatch-provoked Reactions in an Extract System

The mismatch-provoked and control reactions in the extract system were carried out at 37 °C in 80-μl mixtures containing 20 mm HEPES-NaOH (pH 7.4), 100 mm KCl, 8 mm MgCl2, 2 mm DTT, 0.2 mg/ml BSA, 0.1 mm each of the four dNTPs, 3 mm ATP, 20 mm creatine phosphate, 0.02 mg/ml creatine phosphokinase, 1% glycerol (v/v), 150 μg of 293T cell extract, MutLα (0 or 3.2 pmol), MutLα-E705K (0 or 3.2 pmol), purified CAF-1 (0, 0.3, 0.6, or 2.4 pmol), and 41 or 162 fmol of a 3′-nicked DNA (the 3′-nicked O6-mG-T DNA, the 3′-nicked G-T DNA, or the 3′-nicked A-T DNA). The reaction mixtures were incubated for 10–120 min. Reactions in each mixture were stopped by the addition of a 60-μl solution containing 0.35% SDS, 0.4 m NaCl, 13 mm EDTA, 0.33 mg/ml Proteinase K, and 1 mg/ml glycogen, and the resulting mixtures were incubated for 20 min at 50 °C, followed by their extraction with phenol/chloroform mixture. DNAs present in the supernatants were recovered by isopropyl alcohol precipitation. To detect whether the repair of O6-mG-T or G-T mispairs occurred in a reaction mixture, a fraction of the recovered DNA was cleaved with BanI and XhoI and separated on a 1.2% agarose gel, followed by staining of the gel with ethidium bromide and quantification of DNA species with the ImageJ software. To detect whether a 3′-nicked DNA was degraded in the reaction, a fraction of the recovered DNA was cleaved with ClaI, separated on an alkaline 1.4% agarose gel, transferred onto a nylon membrane, and hybridized with a 32P-labeled probe. To detect whether gapped DNA was produced in a reaction mixture, a fraction of the recovered DNA was digested with AhdI, resolved on a native 1.4% agarose gel, transferred onto a nylon membrane, and hybridized with an indicated 32P-labeled probe. Indirectly labeled DNA species were visualized and quantified as described above.

Cleavage of DNA by Activated MutLα Endonuclease in a Defined System

The reactions were performed in 40-μl mixtures that contained 20 mm HEPES-NaOH (pH 7.4), 120 mm KCl, 5 mm MgCl2, 3 mm ATP, 2 mm DTT, 0.2 mg/ml BSA, 2% glycerol (v/v), and 2 nm (81 fmol) of a 3′-nicked DNA (the 3′-nicked O6-mG-T DNA, the 3′-nicked G-T DNA, or the 3′-nicked A-T DNA). When indicated, MutSα (40 nm), MutLα (2, 6, or 20 nm), MutLα-E705K (20 nm), PCNA (24 nm), RFC (4 nm), and RPA (40 nm) were included in the reaction mixtures. The reaction mixtures were incubated for 10 min at 37 °C, and each reaction was terminated by the addition of a 30-μl mixture containing 0.35% SDS, 0.4 m NaCl, 13 mm EDTA, 0.33 mg/ml Proteinase K, and 2 mg/ml glycogen, followed by incubation of the mixtures for 15 min at 50 °C. The mixtures were then extracted by phenol/chloroform, and the DNAs were recovered by isopropyl alcohol precipitation. Recovered DNAs that were digested or not with ClaI were separated on alkaline 1.4% denaturing agarose gels, transferred onto nylon membranes, and hybridized with 32P-labeled probes. Indirectly labeled DNA species were visualized and quantified as described above.

Author Contributions

L. K., B. D., and F. K. performed experiments and analyzed data. F. K. and L. K. designed experiments, contributed reagents, prepared the figures, and wrote the paper.

Acknowledgment

We thank Farid F. Kadyrov for critical reading of the manuscript.

*This work was supported by NIGMS, National Institutes of Health, Grant R01GM095758. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2The abbreviations used are:

MMR
DNA mismatch repair
PCNA
proliferating cell nuclear antigen
RFC
replication factor C
RPA
replication protein A
Pol
DNA polymerase
O6-mG
O6-methylguanine
nt
nucleotide(s).

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