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* This work was supported, in whole or in part, by National Institutes of Health Grant R01GM095758 from NIGMS. The authors declare that they have no conflicts of interest with the contents of this article. ♦ This article was selected as a Paper of the Week.
The DNA mismatch repair (MMR) system plays a major role in promoting genome stability and suppressing carcinogenesis. In this work, we investigated whether the MMR system is involved in Okazaki fragment maturation. We found that in the yeast Saccharomyces cerevisiae, the MMR system and the flap endonuclease Rad27 act in overlapping pathways that protect the nuclear genome from 1-bp insertions. In addition, we determined that purified yeast and human MutSα proteins recognize 1-nucleotide DNA and RNA flaps. In reconstituted human systems, MutSα, proliferating cell nuclear antigen, and replication factor C activate MutLα endonuclease to remove the flaps. ATPase and endonuclease mutants of MutLα are defective in the flap removal. These results suggest that the MMR system contributes to the removal of 1-nucleotide Okazaki fragment flaps.
system promotes genome stability by correcting replicative DNA polymerase errors, removing mismatches formed during homologous recombination, impeding homologous recombination, and participating in DNA damage response (
MutLα (MLH1-PMS2 heterodimer in humans and MLH1-PMS1 heterodimer in yeast), MutSα (MSH2-MSH6 heterodimer), MutSβ (MSH2-MSH3 heterodimer), EXO1, PCNA, and RFC are the key eukaryotic MMR factors (
). Eukaryotic MMR occurs both on the leading and lagging strands, but mismatches on the lagging strands are corrected more efficiently than those on the leading strands (
). After mismatch recognition, MutSα or MutSβ and loaded PCNA activate MutLα to incise the discontinuous daughter strand in the vicinity of the mismatch (
). A strand break generated by MutLα 5′ to the mismatch serves as the entry site for MutSα-activated exonuclease 1 to degrade a mismatch-containing segment of the daughter strand in a 5′ → 3′-excision reaction (
). The reconstituted system bypasses the requirement for exonuclease 1 in the mismatch removal by relying on the strand-displacement activity of DNA polymerase δ holoenzyme.
In addition to mismatches, several other aberrant structures with significant mutagenic potential are formed during DNA replication. Among them are Okazaki fragment flaps (
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
). In eukaryotes, Rad27/FEN1 endonuclease, Dna2 helicase/nuclease, and the 3′ → 5′-exonuclease activity of DNA polymerase δ remove Okazaki fragment flaps (
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
The 3′→5′ exonucleases of DNA polymerases δ and ϵ and the 5′→3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities.
). It has been unknown whether the MMR system plays a direct role in DNA replication. In this report, we describe genetic and biochemical experiments that indicate that the MMR system removes 1-nt Okazaki fragment flaps.
Experimental Procedures
Yeast Strains and Genetic Methods
Saccharomyces cerevisiae wild-type haploid strains used in this study were as follows: FKY688 (MATα ade5-1 lys2::InsE-A14trp1-289 his7-2 leu2-3,112 ura3-52 V29617::URA3) (
). The wild-type diploid strain FKY1037 was prepared by crossing the E134 and 1B-D770 strains. Gene replacements were generated by transforming yeast haploid or diploid cells with disruption cassettes in the presence of lithium acetate/PEG4000/DMSO. The PMS1 gene located in its natural chromosomal location was mutated to the pms1-E707K allele using the “dellitto perfetto” technique (
Human MutSα, MutLα, MutLα-D699N, MutLα-E705K, MutLα-EA, PCNA, RFC, RPA, CAF-1, histone H3-H4 complex, and FEN1 were isolated in nearly homogeneous forms as described previously (
). Yeast MutSα containing the FLAG tag at the N terminus of its Msh6 subunit was expressed in and purified from insect Sf9 cells. The protein that was used in the DNA-binding reactions was more than 95% pure.
Gel Mobility Shift Assays
Gel mobility shift assays that used the oligonucleotide-based substrates (Fig. 2) were carried out as described below. The oligonucleotide-based substrates were produced using oligonucleotides 1–8 (Table 1). Each of the substrates contained oligonucleotide 1, which was labeled with 32P at the 5′ end with T4 polynucleotide kinase. In addition, the homoduplex, 1-nt insertion, dynamic 1-nt DNA flap, static 1-nt 3′ DNA flap, static 1-nt 5′ DNA flap, and nicked substrates contained oligonucleotides 2, 3, 4 and 5, 6 and 7, 4 and 8, and 4 and 7, respectively. To make the DNA substrates, the indicated oligonucleotides were mixed and annealed. The annealing was carried out in a buffer containing 20 mm HEPES-NaOH, pH 7.4, and 100 mm KCl at 40 °C for 4 h, followed by incubation of the mixtures at 20 °C for 30 min. After annealing, the resulting duplex DNAs were separated on native 6% polyacrylamide gels and then purified from the gels. The gel-purified DNAs were used as substrates in the DNA-binding reactions. The DNA-binding reactions were carried out in 20-μl mixtures each containing 20 mm HEPES-NaOH, pH 7.4, 5 mm MgCl2, 140 mm KCl, 0.2 mg/ml BSA, 2 mm DTT, 20 nm of a competitor 40-bp DNA, 2 nm of the indicated 32P-labeled DNA substrate, and purified yeast or human MutSα. Yeast MutSα concentration in the mixtures varied in the range of 5–1600 nm (the actual concentrations used were 5, 10, 20, 40, 100, 200, 400, 550, 800, 1200, and 1600 nm). Human MutSα concentration in the mixtures was in the range of 5–800 nm (the actual concentrations used were 5, 10, 20, 40, 100, 200, 400, 550, and 800 nm). The competitor 40-bp DNA was prepared by annealing two complementary phosphorylated 40-mer oligonucleotides 9 and 10. Reaction mixtures containing yeast MutSα were incubated for 10 min at 30 °C, and reaction mixtures containing human MutSα were incubated for 5 min at 37 °C. The reaction products were immediately subjected to electrophoresis on 6% polyacrylamide gels in the 0.5× Tris borate/EDTA running buffer at 4 °C. The gels were dried, and 32P-labeled DNAs were visualized with a Typhoon phosphorimager (GE Healthcare). Each experiment was repeated at least twice. After quantification of the images with ImageQuant software (GE Healthcare), the apparent Kd values were determined using GraphPad Prism 6 software. The data were fit into the equation of nonlinear regression curve with Hill slope (Y = Bmax·Xh/(Kd + Xh)). In this equation, Y is the concentration of MutSα-DNA complexes; Bmax is the maximum concentration of MutSα-DNA complexes; X is the concentration of MutSα; Kd is the apparent dissociation constant, and h is the Hill coefficient.
FIGURE 2Human and yeast MutSα proteins recognize 1-nt DNA flaps. The gel mobility shift assays with the oligonucleotide-based DNA substrates and calculations of the apparent Kd values were performed as described under “Experimental Procedures.” All six substrates had the same bottom strand. The DNA sequences of the homoduplex and nicked DNA substrates were identical to each other and to the his7-2 sequence, in which the majority of +1 frameshifts are formed. Compared with the top strand of the homoduplex or nicked substrate, the top strands of the flapped and 1-nt insertion substrates each contained an extra nucleotide residue, which was necessary to produce the 1-nt flap or 1-nt insertion. A, representative images showing binding of yeast MutSα to the different DNA substrates. Each DNA-binding reaction was carried out in the mixture containing the indicated concentration of yeast MutSα and the indicated DNA substrate (2 nm). B and C, apparent Kd values for binding of yeast MutSα (B) and human MutSα (C) to the indicated DNA substrates. The apparent Kd values were calculated using the data that were obtained by quantification of images, including those shown in A. The numbers above the bars are the apparent Kd values.
Gel mobility shift assays that used 2-kb circular DNA substrates (Fig. 3) were performed as detailed below. The substrates were prepared using the pSYAH1A plasmid DNA containing a 36-nt gap (
). The no-flap, G-T, 1-nt DNA flap, and 1-nt RNA flap substrates were prepared by annealing the gapped pSYAH1A DNA with oligonucleotides 11, 12, 13, and 14, respectively. The G-T and no-flap substrates each contain two ligatable nicks that are 36 nt apart. Cleavage with restriction endonucleases HindIII and HpyCH4III was utilized to determine what fraction of each of the substrates contains the annealed oligonucleotide. These restriction endonucleases do not cleave DNA within a gap due to the destruction of their sites by the gap. Based on this approach, we determined that ∼95% of each of the circular substrates contained the annealed oligonucleotide.
FIGURE 3Human MutSα recognizes 1-nt DNA and RNA flaps on 2-kb circular DNA molecules.A, diagrams of the 2-kb circular DNAs used in the DNA-binding reactions. Each diagram also shows the relative position of the hybridization probe (bar with an asterisk). The hybridization probe is complementary to the continuous strand. B, apparent Kd values for binding of human MutSα to the indicated circular substrates. The numbers above the bars are the apparent Kd values. The gel mobility shift assays and calculations of the apparent Kd values were carried out as detailed under “Experimental Procedures.”
To determine apparent Kd values for binding of human MutSα to the circular DNAs, the reactions were carried out in 20-μl mixtures each containing 20 mm HEPES-NaOH, pH 7.4, 120 mm KCl, 5 mm MgCl2, 0.2 mm ATP, 0.2 mg/ml BSA, 2 mm DTT, 1.9 nm (50 ng) of the indicated circular 2-kb DNA, 50 nm of the competitor 40-bp DNA, and human MutSα (5, 10, 20, 40, 100, 200, 400, 550, or 800 nm). After a 5-min incubation at 37 °C, each reaction mixture was mixed with 3 μl of loading buffer (1× TAE, 40% glycerol, and 0.02% bromphenol blue), and the reaction products were immediately subjected to electrophoresis on 1.2% agarose gels in 1× TAE at 4 °C, followed by ethidium bromide staining of the gels. The separated DNAs were transferred onto nylon membranes and hybridized with 32P-labeled oligonucleotide 15. The labeled DNAs were visualized with a Typhoon phosphorimager. The data were quantified and analyzed as described above.
DNA Incision Reactions
Circular DNAs were used as substrates in the incision reactions (FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7, FIGURE 8). Each of the substrates was prepared by annealing of an appropriate 5′-phosphorylated or 5′-32P-labeled oligonucleotide to the gapped pSYAH1A DNA in a mixture containing the oligonucleotide and gapped DNA in a 1:1 molar ratio. The diagnostic cleavage with HindIII and HpyCH4III outlined above showed that 92–96% of each of the substrates contained the annealed oligonucleotide. The 5′-32P label was introduced into the oligonucleotides by T4 polynucleotide kinase. The incision reactions were performed in 25–40-μl mixtures each containing 20 mm HEPES-NaOH, pH 7.4, 120 mm KCl, 5 mm MgCl2, 3 mm ATP, 0.2 mg/ml BSA, 2 mm DTT, 1.5 nm (60 fmol) of the indicated DNA substrate, and the indicated human proteins. When MutSα, MutLα, PCNA, RFC, RPA, CAF-1, MutLα-E705K, MutLα-D699N, and MutLα-EA were present in the reaction mixtures, their concentrations were 40, 16, 24, 4, 40, 24, 16, 16, and 16 nm, respectively. Some DNA incision reactions (FIGURE 6, FIGURE 7, FIGURE 8) occurred in the presence of histone H3-H4 heterodimer (22, 44, or 88 nm). The DNA incision reactions were incubated at 37 °C for 10–30 min as indicated. Unless noted otherwise, the reactions were stopped and analyzed as described below. At the specified times, 8- or 11-μl aliquots of the reactions were mixed with 20 μl of a gel-loading buffer containing 90% formamide and 20 mm EDTA. DNA products of the stopped reactions were separated on 15% polyacrylamide gels containing 6 m urea. The gels were dried, and the 32P-labeled DNA species were visualized by phosphorimaging. The data were quantified using ImageQuant software (GE Healthcare).
FIGURE 41-nt DNA and RNA flaps activate MutLα endonuclease to incise the discontinuous strands in the presence of MutSα, PCNA, RFC, and RPA. Each DNA incision reaction was carried out in the mixture containing the indicated human proteins and DNA substrate (1.5 nm). When MutSα, MutLα, MutLα-E705K, PCNA, RFC, and RPA were present in the reaction mixtures, their concentrations were 40, 16, 16, 24, 4, and 40 nm, respectively. After a 10-min incubation, the reactions were stopped by the addition of NaOH and EDTA to the final concentrations of 40 and 5 mm, respectively. The reaction products were separated on alkaline 1.2% agarose gels, transferred onto nylon membranes, hybridized with 32P-labeled oligonucleotide 16, and visualized by phosphorimaging. A, representative images showing incision of the discontinuous strands in the presence of MutLα, MutSα, PCNA, RFC, and RPA. The diagrams outline the circular DNA substrates. Each diagram also shows the relative position of the hybridization probe (bar with an asterisk). The hybridization probe is complementary to the discontinuous strand. B, summary of incision of the discontinuous strands of the indicated DNA substrates at sites that are 4-nt 3′ to the flap or control nick. The data were obtained by quantification of images, including those shown in A, and are presented as averages ± 1 S.D., n ≥3.
FIGURE 5MutLα endonuclease incises the discontinuous strand four nucleotides downstream from a 1-nt DNA or RNA flap. The 37-nt fragments of the 1-nt DNA and RNA flap-containing substrates and the 36-nt fragments of the control flap-free and G-T substrates were labeled at their 5′ ends with 32P. Each DNA incision reaction was performed in the mixture containing the indicated human proteins and 32P-labeled DNA substrate (1.5 nm). When MutSα, MutLα, MutLα-E705K, PCNA, RFC, and RPA were present in the reaction mixtures, their concentrations were 40, 16, 16, 24, 4, and 40 nm, respectively. The DNA incision reactions were stopped and analyzed as described under “Experimental Procedures.” A, representative image showing MutLα endonuclease-dependent incision of the discontinuous strand 4 nt downstream from the 1-nt flap. The incision reactions were incubated for 10 min. The diagrams outline the circular DNA substrates. B, summary of incision of the discontinuous strands of the indicated substrates at sites that are 4-nt 3′ to the flap or control nick. The DNA incision reactions were incubated for 10 min. C, time course of incision of the discontinuous strands of the indicated substrates at sites that are 4-nt 3′ to the flap or control nick. The incision reactions were carried out in the mixtures containing MutSα (40 nm), MutLα (16 nm), PCNA (24 nm), RFC (4 nm), RPA (40 nm), and the indicated DNA substrate (1.5 nm). The data in B and C are averages ± 1 S.D. (B, n ≥4; C, n ≥3) and were obtained by quantification of images, including the one shown in A.
FIGURE 6CAF-1-dependent histone H3-H4 deposition stimulates the removal of 1-nt flaps by the activated MutLα endonuclease. The 37-nt fragment of the 1-nt DNA flap-containing substrate and the 36-nt fragment of the control flap-free substrate were labeled at their 5′ ends with 32P. Each DNA incision reaction was performed in the mixture containing the indicated human proteins and 32P-labeled DNA substrate (1.5 nm). When MutSα, MutLα, MutLα-D699N, MutLα-EA, PCNA, RFC, RPA, CAF-1, and the histone H3-H4 heterodimer were present in the reaction mixtures, their concentrations were 40, 16, 16, 16, 24, 4, 40, 24, and 88 nm, respectively. The reactions were incubated for 30 min and then stopped and analyzed as described under “Experimental Procedures.” A, representative image showing the effects of the indicated protein combinations on incision of the discontinuous strands of the indicated substrates at sites that are 4-nt 3′ from the flap or control nick. The diagrams outline the circular DNA substrates. B, graphical representation of the effects of the indicated protein combinations on incision of the discontinuous strands of the indicated substrates at sites that are 4-nt 3′ from the flap or control nick. The data were obtained by quantification of images, including the one shown in A and are averages ± 1 S.D., n ≥4. C, dependence of the incision on the presence of the 1-nt DNA flap. The flap dependence values were calculated from the data shown in B. The presence of a statistically significant difference between the flap dependences of the two indicated reactions was identified by unpaired t test.
FIGURE 7CAF-1-dependent histone H3-H4 deposition protects the remote sites from incision by MutLα endonuclease. Each DNA incision reaction was performed in the mixture containing the indicated human proteins and DNA substrate (1.5 nm). When MutSα, MutLα, PCNA, RFC, RPA, and CAF-1 were present in the reaction mixtures, their concentrations were 40, 16, 24, 4, 40, and 24 nm, respectively. After a 30-min incubation, the incision reactions were stopped and analyzed as described in Fig. 4. A, image showing the effects of the different protein combinations on incision of the discontinuous strands of the 1-nt flap-containing and flap-free DNA substrates. The diagrams outline the DNA substrates. Each diagram also shows the relative position of the hybridization probe (bar with an asterisk), which is complementary to the discontinuous strand. B and C, incision of the discontinuous strands of the 1-nt flap-containing and flap-free DNA substrates as a function of concentration of histone H3-H4 heterodimers. The data were obtained by quantification of images, including the one shown in A, and are presented as averages ± 1 S.D., n = 2.
FIGURE 8Flap removal in a reconstituted human system containing FEN1 and MutLα endonucleases. The DNA incision reactions were carried out in the mixtures containing the indicated human proteins and 32P-labeled circular DNA substrate (1.5 nm). When MutSα, MutLα, PCNA, RFC, RPA, CAF-1, and the histone H3-H4 heterodimer were present in the reaction mixtures, their concentrations were 40, 16, 24, 4, 40, 24, and 88 nm, respectively. After incubation for 10 min, the DNA incision reactions were stopped and analyzed as described under “Experimental Procedures.” A, representative image showing the effects of the different protein combinations on the removal of the 1-nt DNA flaps. The arrows indicate the positions of the 1- and 5-nt cleavage products generated by FEN1 and MutLα, respectively. The diagram outlines the circular DNA substrate. B, graphical representation of the effects of the different FEN1 concentrations on the yield of the product of MutLα endonuclease-dependent flap removal in the eight-protein system. The eight-protein system contained MutLα (16 nm), MutSα (40 nm), PCNA (24 nm), RFC (4 nm), RPA (40 nm), CAF-1 (24 nm), histone H3-H4 heterodimer (88 nm), and FEN1 (0.3, 0.6, 1.2, or 2.4 nm). C, graphical representation of the effects of the different FEN1 concentrations on the yield of the product of FEN1-dependent flap removal in the one-protein and eight-protein systems. The one-protein system contained FEN1 (0.3, 0.6, 1.2, or 2.4 nm). The data in B and C were obtained by quantification of images, including the one shown in A and are averages ± 1 S.D., n ≥4.
MMR System and Rad27 Flap Endonuclease Have Overlapping Functions Involved in the Maintenance of Genome Stability
We began this work to investigate whether the MMR system contributes to the removal of Okazaki fragment flaps. The Rad27/FEN1 endonuclease is the key enzyme that removes short flaps during Okazaki fragment maturation (
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
). Previous research has demonstrated that both the MMR system and Rad27 are necessary for the suppression of mutations in the +1 frameshift reporter his7-2 (
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
The 3′→5′ exonucleases of DNA polymerases δ and ϵ and the 5′→3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
). To study whether there is a functional overlap between the MMR system and the Rad27 flap endonuclease, we determined the his7-2 mutation rates in the haploid and diploid yeast strains shown in TABLE 2, TABLE 3. The his7-2 mutation rate in the haploid double mutant msh2Δ rad27Δ (6700 × 10−8) was 33 times higher than the sum of the his7-2 mutation rates in the haploid single mutants msh2Δ and rad27Δ (i.e. combining msh2Δ with rad27Δ resulted in a 33-fold synergistic increase in the his7-2 mutation rate) (Table 2). Likewise, the his7-2 mutation rate for the diploid double mutant msh2Δ/msh2Δ rad27Δ/rad27Δ (13,000 × 10−8) was increased 36 times relative to the sum of the his7-2 mutation rates for the diploid single mutants msh2Δ/msh2Δ RAD27/RAD27 and MSH2/MSH2 rad27Δ/rad27Δ (Table 3). These findings indicate that there is a functional overlap between the MMR system and Rad27 in haploid and diploid yeast S. cerevisiae.
TABLE 2Impact of deletion of MSH2 and RAD27 on rates of his7-2 mutations
). To ascertain that the above findings (TABLE 2, TABLE 3) were not reporter-specific, we measured the lys2::InsE-A8 mutation rates in the msh2Δ, rad27Δ, and msh2Δ rad27Δ mutants (Table 4). Analysis of the data demonstrated that the lys2::InsE-A8 mutation rate in the msh2Δ rad27Δ double mutant (21,000 × 10−8) was 24 times higher than the sum of the lys2::InsE-A8 mutation rates in the msh2Δ and rad27Δ single mutants. Thus, the use of the lys2::InsE-A8 mutation assay provided additional evidence that a genetic stabilization function of the MMR system overlaps with a genetic stabilization function of the Rad27 flap endonuclease. Collectively, these genetic experiments suggest that an MMR system-dependent mechanism and a different mechanism dependent on the Rad27 flap endonuclease repair the same or related types of pre-mutagenic intermediates which, if left unrepaired, give rise to +1 frameshifts.
TABLE 4Effect of combining msh2Δ and rad27Δ on lys2::InsE-A8 mutation rate
Next, we used DNA sequencing to identify +1 frameshifts that reverted his7-2 in the msh2Δ, rad27Δ, and msh2Δ rad27Δ mutants (Table 2). The results revealed that all of the his7-2 reversions in the msh2Δ and msh2Δ rad27Δ spectra and a majority of the reversions in the rad27Δ spectrum were 1-bp insertions, each of which extended the A7 run into an A8 run (Table 2). In addition, we found that combining msh2Δ with rad27Δ led to a 40-fold synergistic increase in the rate of 1-bp insertions (Table 2). This finding implies that one or several related types of pre-mutagenic intermediates producing 1-bp insertions are repaired by both an MMR system-dependent mechanism and a Rad27-dependent mechanism.
The MMR system contains two mismatch recognition complexes, MutSα and MutSβ. As shown in Table 5, the his7-2 mutation rate in the msh3Δ msh6Δ mutant was indistinguishable from that in the msh2Δ mutant but 23 times higher than the sum of those in the msh3Δ and msh6Δ mutants. This result indicates that the partially overlapping activities of MutSα and MutSβ (
) are engaged in the suppression of +1 frameshifts in his7-2. To study whether an MMR system-dependent function overlapping with a Rad27 function involves MutSα and/or MutSβ, we determined the his7-2 mutation rates for the msh2Δ, rad27Δ, msh2Δ rad27Δ, msh3Δ msh6Δ rad27Δ, msh3Δ rad27Δ, and msh6Δ rad27Δ mutants (Table 5). We found that the his7-2 mutation rate for the msh3Δ msh6Δ rad27Δ mutant did not differ from the his7-2 mutation rate for the msh2Δ rad27Δ mutant, but it was ∼12 or ∼70 times higher than the rate for the msh6Δ rad27Δ or msh3Δ rad27Δ mutants, respectively. These data indicate that both MutSα and MutSβ participate in an MMR system-dependent function that overlaps with a Rad27 function. We also found that the his7-2 mutation rate in msh6Δ rad27Δ exceeded that in msh3Δ rad27Δ by 6-fold (Table 5). This result is consistent with the view that compared with MutSβ, MutSα plays a more important role in an MMR system-dependent function that overlaps with a Rad27 function.
TABLE 5Effects of the different mutant combinations on his7-2 mutation rate
). The endonuclease activity of yMutLα depends on the integrity of the Pms1 DQHA(X)2E(X)4E motif, which is part of the putative activesite of the endonuclease (
). We found that combining rad27Δ with mlh1Δ, pms1Δ, or pms1-E707K resulted in a 20–26 times synergistic increase in the his7-2 mutation rate (Table 5). Nevertheless, the his7-2 mutation rate in the pms1-E707K rad27Δ, pms1Δ rad27Δ, or mlh1Δ rad27Δ strain was half that in the msh2Δ rad27Δ strain (Table 5). Taken together, these data suggest that an MMR system-dependent function overlapping with a Rad27 function often involves the endonuclease activity of MutLα.
The results described above were obtained using the his7-2 and lys2::InsE-A8 reversion assays that only allow scoring of +1 frameshifts. Unlike the his7-2 and lys2::InsE-A8 reversion assays, the CAN1 forward mutation assay allows scoring of many different types of genetic alterations, including 1-bp insertions, base substitutions, and 1-bp deletions. The CAN1 forward mutation assay takes advantage of the fact that mutational inactivation of the CAN1 gene encoding arginine permease makes the yeast cell resistant to canavanine, a structural analog of arginine. In this assay, Canr cells are selected on a synthetic medium that lacks arginine and contains canavanine. To determine the can1 mutation spectrum in an msh2Δ rad27Δ strain, we performed a series of experiments summarized in Fig. 1. We started this series of experiments by measuring the CAN1 mutation rates in two sets of msh2Δ, rad27Δ, and msh2Δ rad27Δ strains (Fig. 1A). One set of the strains was prepared on the wild-type strain E134 background and the other on the wild-type strain BY4742 background. We chose to measure CAN1 mutation rates in two sets of yeast strains to exclude the possibility that the data are strain-specific. The results demonstrated that the relative CAN1 mutation rate in either msh2Δ rad27Δ mutant was ∼2 times higher than the sum of the relative CAN1 mutation rates in the isogenic single mutants (i.e. the relative CAN1 mutation rates in the isogenic msh2Δ and rad27Δ mutants are in a weak synergistic relationship) (Fig. 1A). Similar results were obtained in two earlier studies (
). We next determined the can1 mutation spectra in the wild-type, msh2Δ, rad27Δ, and msh2Δ rad27Δ mutants (Fig. 1B). The rates of base substitutions and 1-nt deletions in the msh2Δ rad27Δ mutant did not differ significantly from those in the msh2Δ mutant. However, the rate of 1-nt insertions in the msh2Δ rad27Δ mutant was 12 times higher than sum of those in the msh2Δ and rad27Δ mutants. This information supports the view that one or several related types of pre-mutagenic intermediates causing 1-nt insertions are removed by both an MMR system-dependent mechanism and a Rad27-dependent mechanism.
FIGURE 1CAN1 mutation rates and can1 mutation spectra in the wild-type, msh2Δ, rad27Δ, and msh2Δ rad27Δ strains.A, CAN1 mutation rates. Each of the mutants was made in the two different wild-type backgrounds: E134 and BY4742. The numbers above the bars are the relative mutation rates. B, can1 mutation spectra in the wild-type strain E134 and its mutant derivatives. The relative mutation rates are in parentheses. a, all 1-bp insertions were formed in mononucleotide runs that were ≥N2.
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
). Strikingly, 6–14-bp duplications were produced at a rate of 630 × 10−8 in CAN1 in the msh2Δ rad27Δ strain, but they were absent in the can1 spectra of the rad27Δ and msh2Δ mutants (Fig. 1B). These data suggest that one or several related types of pre-mutagenic intermediates triggering 6–14-bp duplications are removed by both an MMR-dependent mechanism and a Rad27-dependent mechanism.
The Dna2 helicase/nuclease is an essential enzyme that participates in the removal of flaps during Okazaki fragment maturation (
Dna2 mutants reveal interactions with Dna polymerase α and Ctf4, a pol α accessory factor, and show that full Dna2 helicase activity is not essential for growth.
). We established that the his7-2 mutation rate in the dna2-1 strain was increased 10-fold relative to that in the wild-type strain (Table 6). Sequencing of 10 independent HIS7 revertants produced in the dna2-1 background showed that nine mutants contained an identical mutation, which was an A insertion in the his7-2 A7 run, and one mutant had a deletion of two As in the same run. We then studied the effect of combining dna2-1 with msh2Δ on the his7-2 mutation rate (Table 6). We found that the his7-2 mutation rate in the dna2-1 msh2Δ double mutant was two times higher than the sum of those in the single mutants. This observation is consistent with the idea that one or several related types of pre-mutagenic intermediates causing +1 frameshifts are repaired by both an MMR system-dependent mechanism and a Dna2-dependent mechanism.
TABLE 6Effect of dna2-1 msh2Δ on his7-2 mutation rate
We considered two models to explain the observation that combining msh2Δ with rad27Δ leads to the strong synergistic increases in the rates of spontaneous 1-bp insertions (Table 2 and Fig. 1B). In the first model, DNA polymerase α errors are corrected not only by MMR (
) but also by a Rad27-dependent mechanism, and DNA polymerase α errors that escape both MMR and the Rad27-dependent mechanism produce mutations, including 1-bp insertions. However, this model is not supported by the observation that the deletion of RAD27 in the msh2Δ strain does not significantly increase the rate of base substitutions (Fig. 1B), which are the most common products of DNA polymerase α errors (
). Thus, it is unlikely that a considerable fraction of 1-bp insertions formed in msh2Δ rad27Δ mutants originate from DNA polymerase α errors. The second model is based on the knowledge that the key function of the 5′ flap endonuclease Rad27 is the removal of short Okazaki fragment flaps (
). In this model, 1-nt Okazaki fragment flaps are removed by both a Rad27-dependent mechanism and an MMR system-dependent mechanism, and the unprocessed flaps are converted by misalignment and ligation into 1-bp insertions. Thus, this model suggests that the majority of 1-bp insertions produced in msh2Δ rad27Δ mutants are formed from 1-nt Okazaki fragment flaps. Because Okazaki fragment flaps do not cause base substitutions, the second model is consistent with our genetic data (Table 2 and Fig. 1B).
The second model postulates that the MMR system removes 1-nt Okazaki fragment flaps. To determine whether there is evidence for this, we carried out the biochemical experiments described below. We first examined whether yeast MutSα recognizes 1-nt DNA flaps present on the 32P-labeled oligonucleotide-based substrates (Fig. 2). The data revealed that yeast MutSα bound the substrate containing the dynamic 1-nt flap with an apparent Kd of 38 ± 2 nm (Fig. 2, A and B). The control experiments indicated that yeast MutSα bound the 1-nt insertion-containing DNA, nicked DNA, and homoduplex DNA with apparent Kd values of 25 ± 1, 180 ± 10, and 200 ± 8 nm, respectively (Fig. 2, A and B). Therefore, these experiments demonstrate that yeast MutSα recognizes the dynamic 1-nt flap nearly as efficiently as the 1-nt insertion. We then investigated whether yeast MutSα recognizes static 1-nt 3′ and 5′ flaps. The experiments showed that yeast MutSα bound the static 1-nt 3′ and 5′ flaps with apparent Kd values of 60 ± 2 and 55 ± 3 nm, respectively. Thus, yeast MutSα recognizes the static 1-nt 3′ and 5′ flaps with the same affinity. Surprisingly, yeast MutSα detected the static 1-nt 3′ and 5′ flaps somewhat less efficiently than the dynamic 1-nt flap (Fig. 2, A and B). Because a dAMP residue forms the flap in the dynamic substrate and a dCMP residue produces the flaps in the static substrates, it is possible that yeast MutSα recognizes a flapped dCMP residue less efficiently than a flapped dAMP residue.
We also studied whether human MutSα recognizes the dynamic 1-nt flap (Fig. 2C). Our experiments indicated that human MutSα bound the dynamic 1-nt flap with an apparent Kd of 30 ± 1 nm. An apparent Kd value for binding of human MutSα to the 1-nt insertion is 30 ± 6 nm. These Kd values are 7–12 times lower than those for binding of human MutSα to the nicked and homoduplex DNAs (Fig. 2C). Thus, human MutSα efficiently recognizes the dynamic 1-nt flap. Collectively, these findings support the view that the ability to recognize 1-nt DNA flaps is conserved in eukaryotic MutSα proteins.
We also analyzed whether human MutSα recognizes a dynamic 1-nt flap present on a circular 2-kb DNA (Fig. 3). Each of the substrates contained a 1-nt DNA flap, a 1-nt RNA flap, no flap, or a G-T mispair (Fig. 3A). The results revealed that MutSα bound the 1-nt DNA and RNA flap-containing DNAs with Kd values of 119 ± 3 and 115 ± 10 nm, respectively (Fig. 3B). These Kd values are half that of 254 ± 35 nm for the binding of MutSα to the control no-flap DNA. Thus, MutSα detects that the circular DNA carries a 1-nt flap, which may be a deoxyribonucleotide or ribonucleotide residue.
MutLα Endonuclease-dependent Removal of 1-nt Flaps
Having shown that MutSα recognizes the 1-nt DNA and RNA flaps on the circular DNA, we carried out and analyzed the reconstituted reactions to determine whether these flaps activate human MutLα endonuclease to incise the discontinuous strand in the presence of human MutSα, PCNA, RFC, and RPA (Fig. 4). The circular DNAs were used as substrates in these reactions because loaded PCNA, required for the activation of MutLα endonuclease (
), slides off of linear DNA. The reactions were performed under conditions that were very similar to those used for the identification of the MutSα-, PCNA-, RFC-, mismatch-, and ATP-dependent endonuclease activity of human MutLα (
). Analysis of the reactions (Fig. 4, A and B) led to the following observations. First, 34 ± 5% of the discontinuous strand of the 1-nt DNA flap-containing substrate was incised by MutLα, whereas the endonuclease cleaved only 10 ± 2% of the discontinuous strand of the control flap-free substrate. Second, MutLα incised 30 ± 1% of the discontinuous strand of the 1-nt RNA flap. Third, an endonuclease-deficient MutLα variant, MutLα-E705K (
), did not incise the discontinuous strands of the tested substrates. Together, these observations indicate that 1-nt flaps activate MutLα endonuclease to incise the discontinuous strand in the presence of MutSα, PCNA, RFC, and RPA.
To determine whether incision of the discontinuous strand by MutLα results in the removal of flaps, we performed experiments summarized in Fig. 5. As shown in lane 2 of Fig. 5A, the incubation of MutLα, MutSα, PCNA, RFC, and RPA with the 1-nt DNA flap-containing circular substrate led to incision of the 32P-labeled 37-nt fragment at several sites. The most abundant product of the incision reaction had an apparent length of 5 nt, indicating that the incision occurred at a site that is four nucleotides 3′ to the flap. The incision products were not formed when MutSα, MutLα, RFC, or PCNA was omitted from the reaction mixture, but the omission of RPA did not have a significant effect on the incision (Fig. 5, A, lanes 3, 4, 6, and 7, and B). These results indicate that MutSα, MutLα, RFC, and PCNA are required for the incision, but RPA is not. The time course experiments demonstrated that the incision reaction produced the 5-nt fragment in a time-dependent manner (Fig. 5C). The efficiency of the incision of the site located 4 nt downstream from a 1-nt flap was three times higher than that of the same site on the control flap-free substrate (Fig. 5, A, lanes 2 and 10, and B and C). Thus, the flap dependence of the MutLα incision was 3-fold. Changing the incubation temperature from 37 to 25 °C decreased the flap dependence of the MutLα incision from 3- to 2-fold (data not shown). MutLα, MutSα, PCNA, and RFC were also required for the incision of the 1-nt RNA flap-containing substrate (Fig. 5, A, lanes 18–20, 22, and 23, and B). Consistent with a previous study (
), the 5-nt incision product containing the 5′-ribonucleotide residue migrated in the gel slightly slower than the 5-nt incision product lacking a ribonucleotide residue (Fig. 5A, lanes 2 and 18).
We also studied whether the endonuclease activity of MutLα is necessary for the incision of the discontinuous strand at a 1-nt flap (Fig. 5, A and B). The replacement of the wild-type MutLα with the endonuclease-deficient MutLα-E705K led to the disappearance of the incision products indicating that the endonuclease activity of MutLα is responsible for the incisions (Fig. 5, A, lanes 5 and 21, and B). Further analysis revealed that the presence of a 1-nt flap did not activate the MutLα endonuclease to incise the discontinuous strand immediately upstream from the flap (data not shown). Taken together, these experiments demonstrate that MutSα, RFC, and PCNA activate MutLα endonuclease to incise the discontinuous strand 4 nt downstream from a 1-nt DNA or RNA flap. Because the incision is so close to the flaps, it triggers their dissociation from the substrates.
Newly replicated DNA is rapidly assembled into nucleosomes by a mechanism that depends on the histone H3-H4 chaperone CAF-1 (
). The first step in CAF-1-dependent nucleosome assembly is the deposition of histone H3-H4 tetramers. CAF-1-dependent nucleosome assembly probably impacts many processes that take place on the nascent DNA. Consistent with this idea, CAF-1-dependent nucleosome assembly modulates MMR (
). Because the MMR system-dependent flap removal (Fig. 5) is likely to occur during CAF-1-dependent nucleosome assembly, we studied whether histone H3-H4 deposition by CAF-1 affects the flap-removing activity of the MMR system. We determined that CAF-1-dependent histone H3-H4 deposition stimulated the flap-removing activity of the MMR system by 2-fold (Fig. 6, A, lanes 11 and 12, and B) and increased the flap dependence of the incision from 3- to 6-fold (Fig. 6C). The efficiency of the flap removal was not changed when MutSα and MutLα were added to the reaction mixtures that were incubated with CAF-1, the histone H3-H4 complex, PCNA, RFC, and RPA for 15 min suggesting that the MMR system efficiently removes 1-nt DNA flaps in the presence of pre-loaded H3-H4 tetramers (data not shown). The omission of CAF-1 significantly decreased both the efficiency and flap dependence of the incision (Fig. 6C). Control experiments revealed that the flap removal occurring in the presence of CAF-1-dependent histone H3-H4 deposition required both MutSα and MutLα (Fig. 6, A and B). An endonuclease-deficient MutLα variant, MutLα-D699N (
Mutations within the hMLH1 and hPMS2 subunits of the human MutLα mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSα.
), could not substitute for the wild-type MutLα in the incision reaction. Thus, these experiments demonstrate that the CAF-1-dependent histone H3-H4 deposition promotes the removal of 1-nt DNA flaps by the activated MutLα endonuclease.
We then studied how CAF-1 and the histone H3-H4 complex affect the incision of the discontinuous strand at sites that are distant from the 1-nt flap (Fig. 7). Strikingly, the presence of CAF-1 and the histone H3-H4 complex suppressed the MutLα endonuclease-dependent incision of the discontinuous strand at the remote sites (Fig. 7, A, lanes 9 and 14–16, and B). A similar suppression of the MutLα endonuclease-dependent incision of the discontinuous strand was observed in the six-protein system containing the histone H3-H4 complex but not CAF-1 (Fig. 7, A, lanes 9–12, and B). These findings imply that both CAF-1-dependent histone H3-H4 deposition onto the DNA and nonspecific binding of the histone H3-H4 complex to the DNA protect the remote sites from the incision by the activated MutLα endonuclease.
Next, we performed experiments to study whether the reconstituted MMR system is able to remove flaps in the presence of FEN1 (Fig. 8). The data showed that increasing the FEN1 concentration decreased the yield of the product of MutLα endonuclease-dependent flap removal and increased the yield of the product of FEN1-dependent flap removal (Fig. 8, A–C). In addition, the data indicated that one or several proteins present in the eight-protein system suppressed the flap endonuclease activity of FEN1 (Fig. 8, A, lanes 3–10, and C). These experiments provide evidence that the MMR system removes flaps in the presence of FEN1 and suggest that the flap endonuclease activities of FEN1 and the MMR system compete with each other.
Discussion
High fidelity DNA replication is required for the maintenance of genome integrity and the suppression of human diseases (
). We have used genetic analysis and reconstituted systems to study whether the MMR system contributes to the removal of Okazaki fragment flaps. The major findings described in this report are: 1) combining rad27Δ with msh2Δ produces strong synergistic increases in the rates of 1-bp insertions in his7-2 and CAN1 (Table 2 and Fig. 1B); 2) combining rad27Δ with mlh1Δ, pms1Δ, or pms1-E707K causes a 20–26 times synergistic increase in the rate of +1 frameshifts in his7-2 (Table 5); 3) purified yeast and human MutSα proteins recognize 1-nt flaps (FIGURE 2, FIGURE 3); 4) MutLα endonuclease activated by MutSα, RFC, and PCNA removes 1-nt flaps (Fig. 5); 5) the flap-removing activity of the reconstituted MMR system is stimulated by CAF-1-dependent histone H3-H4 deposition (Fig. 6); and 6) the reconstituted MMR system removes 1-nt flaps in the presence of FEN1 (Fig. 8).
These findings indicate that the eukaryotic MMR system removes a subset of 1-nt Okazaki fragment flaps and support a model illustrated in Fig. 9. This model suggests that MutSα, MutLα, PCNA, and RFC provide the minimal set of activities required for the removal of 1-nt Okazaki fragment flaps by the MMR system. According to this model, the mechanism of the removal of a 1-nt Okazaki fragment flap by the MMR system can be divided into three key steps as follows: recognition of the flap by MutSα; activation of MutLα endonuclease by MutSα, PCNA, and RFC; and the removal of the flap by the activated MutLα endonuclease. Our genetic results also suggest that there is an Msh2-dependent, MutLα-independent mechanism of removal of 1-nt Okazaki fragment flaps (Table 5). In addition, our genetic results are compatible with another model. In this model, misalignment and ligation converts some 1-nt Okazaki fragment flaps into 1-nt loops, which are then removed by the strand-specific MMR (
). However, it has not yet been demonstrated that a replicative DNA ligase is able to convert 1-nt flaps into 1-nt loops in the presence of Rad27/FEN1 and/or the MMR system.
FIGURE 9Role for the MMR system in DNA replication. The model suggests that the MMR system supports DNA replication by removing 1-nt Okazaki fragment flaps. The process of the removal of a 1-nt Okazaki fragment flap by the MMR system is initiated by the recognition of the flap by MutSα. In the next step, MutSα acts in conjunction with PCNA and RFC to activate MutLα endonuclease. The activated MutLα endonuclease then removes the flap.
The absolute his7-2 mutation rate in the rad27Δ/rad27Δ msh2Δ/msh2Δ diploid (Table 3) is half that of the previously described strong mutator diploid pol3-01/pol3-01 msh2Δ/msh2Δ (
The 3′→5′ exonucleases of DNA polymerases δ and ϵ and the 5′→3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
). (The pol3-01 mutation inactivates the proofreading activity of DNA polymerase δ.) This observation reveals that the MMR system is nearly as important for the removal of +1 frameshift intermediates in rad27Δ strains as for the repair of +1 frameshift intermediates in pol3-01 strains. Genetic interactions between the MMR system and Rad27 have been investigated in the past (
), but none of the previous studies utilized a +1 frameshift assay or determined can1 mutation spectrum in a strain that lacks an MMR gene and RAD27. Nevertheless, Johnson et al. (
) reported that the relative CAN1 mutation rate in the msh2Δ mutant is in a weak synergistic relationship with that in the rad27Δ mutant. Thus, the results of the measurements of the relative CAN1 mutation rates in the msh2Δ, rad27Δ, and msh2Δ rad27Δ mutants obtained in this work (Fig. 1A) and the study of Johnson et al. (
). We have described in this report that MutSα recognizes 1-nt DNA/RNA flaps (FIGURE 2, FIGURE 3). This finding extends the range of potentially mutagenic DNA structures recognized by MutSα. Our genetic experiments support the idea that MutSβ plays a role in the MMR system-dependent removal of 1-nt Okazaki fragment flaps (Table 5). Thus, it is possible that MutSβ, like MutSα, recognizes 1-nt DNA/RNA flaps and activates MutLα endonuclease to remove them. This would be in line with previous work that identified that MutSβ specifically binds a variety of DNA recombination structures, including the noncomplementary 5′ DNA flaps and 3′ tails (
In the crystal structure of MutSα·G-T DNA complex, the Glu-434 residue of the conserved mismatch recognition FXE motif forms a hydrogen bond with the mispaired T, the conserved Phe-432 residue stacks onto the T, DNA is sharply bent at the mismatch, and there are several nonspecific protein-DNA interactions (
). The intrinsic bendability of duplex DNA at a mismatch is thought to strongly contribute to the recognition of the mismatch by MutS. A recent study has shown that the same mechanism of mismatch recognition is employed by MutSα (
). We speculate that the MSH6 FXE motif is responsible for the recognition of flaps by MutSα. If this is the case, the conserved Glu-434 is a strong candidate to interact with a flapped deoxy- or ribonucleotide residue via a hydrogen bond. It has been described that duplex DNA bends at nicks (
). Therefore, DNA bending at a nick that accompanies the flap may facilitate the flap recognition by MutSα. Because the MMR system is conserved from bacteria to humans (
), it is possible that the MMR system also contributes to the removal of Okazaki fragment flaps in bacteria.
Eukaryotic DNA transactions occur in the nucleosomal environment. The fact that the size of naked nascent DNA strands at a eukaryotic replication fork is only ∼450 bp (
). Our analysis demonstrates that the CAF-1-dependent histone H3-H4 deposition increases the efficiency and specificity of the flap removal by MutLα and protects the discontinuous strand from MutLα incision at the remote sites (FIGURE 6, FIGURE 7). The mechanism behind these effects is not known. We speculate that the loaded histones H3-H4 tetramers trap the MutLα-containing incision complex at the flap-containing site where it was assembled, and as a result the MutLα is not able to incise the discontinuous strand at the remote sites and instead removes the flap.
Previous research demonstrated that during eukaryotic Okazaki fragment maturation, the strand displacement activity of DNA polymerase δ (
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
). In this report, we have described evidence that the eukaryotic MMR system contributes to the removal of Okazaki fragment flaps.
Author Contributions
F. A. K. and L. Y. K. designed experiments. L. Y. K., B. K. D., and F. A. K. performed experiments and analyzed data. F. A. K. and L. Y. K. wrote the paper.
Acknowledgments
We are grateful to Paul Modrich for advice and insightful discussions; Farid F. Kadyrov for critical reading of the manuscript; and Francesca Storici and Kirill Lobachev for help with the dellitto perfetto technique. We thank Tim Formosa for supplying the dna2-1 strain and Paul Modrich and Mike Resnick for providing the plasmids used in this work.
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability.
The 3′→5′ exonucleases of DNA polymerases δ and ϵ and the 5′→3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities.
Dna2 mutants reveal interactions with Dna polymerase α and Ctf4, a pol α accessory factor, and show that full Dna2 helicase activity is not essential for growth.
Mutations within the hMLH1 and hPMS2 subunits of the human MutLα mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSα.
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