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. 2007 Oct;189(19):6976-88.
doi: 10.1128/JB.00776-07. Epub 2007 Jul 6.

The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications

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The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications

Ella Rotman et al. J Bacteriol. 2007 Oct.

Abstract

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT-->CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our DeltamutT allele elevates the AT-->CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT-->CG transversions in our mutT(+) progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in DeltamutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the DeltamutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation.

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Figures

FIG. 1.
FIG. 1.
8-Oxo-guanine, its alternative pairing schemes, as well as the Mut proteins that counteract potential mutagenic consequences of 8-oxo-G (OG). (A) The structure of guanine compared to that of 8-oxo-guanine. (B) 8-Oxo-G · C pair (both in the anti conformation) versus 8-oxo-G · A pair (8-oxo-G is in the syn conformation). (C) The current understanding of the 8-oxo-G mutation-avoidance pathways (after references , , and 73). The top pathway leads toward the GC→TA transversion through A incorporation across template 8-oxo-G and is counteracted by MutM and MutY. Note that besides DNA-guanine oxidation, 8-oxo-guanine incorporation opposite the correct C residue without subsequent excision should also cause an increase in the GC→TA transversion down the line, as the template 8-oxo-G invites misincorporation of the A residue. The middle pathway shows 8-oxo-dGTP interception by MutT. The bottom pathway shows the AT→CG transversion through 8-oxo-G incorporation across the A residue in the template DNA and its enhancement by MutY. The table shows published data (27, 78) that were normalized to the wild type and rounded up to illustrate the logic of the scheme.
FIG. 2.
FIG. 2.
Do the mutT mutants depend on double-strand break repair and induce SOS? (A) A scheme of the assumed replication fork collapse as a result of 8-oxo-guanine (OG) excision in the template DNA in front of the fork. The same logic applies for the rdgB mutants, only 8-oxo-guanine is replaced with hypoxanthine. (B) Viability of mutT rec double and triple mutants at the 42°C temperature, nonpermissive for the rec alleles. A total of 10 μl of 10−4 dilutions of rapidly growing cultures was spotted on LB plates and incubated either at 27°C for 36 h or at 42°C for 20 h. The ΔrecA304 rdgB-17 p(ori-Ts) recA+ strain (44) is included as a positive control for the Rec dependence. The strains are as follows: AB1157, the wild-type strain; rdgB-17 ΔrecA304, EL002; rdgB-17, EL003; ΔmutT ΔruvABC, ER6; ΔmutT ΔrecA304, ER5; ΔmutT recA200(Ts), ER4; ΔmutT recBC(Ts), ER3; ΔmutT, ER2. (C) The level of SOS induction in ΔmutT mutant cultures. The seqA mutant is shown as an example of the level of SOS induction in RecA-dependent mutants (44). The strains are as follows: wild type, AK43; wild type+MC, AK43 grown in the presence of 100 ng/ml mitomycin C as a control for SOS induction (44); ΔrecA, ER65; ΔmutT, ER27; ΔseqA, ER26. The values are means ± standard error of the mean (for recA, n = 3; for others, n = 6 to 10). The SOS level in the mutT mutant is significantly different from the one in wild-type cells (t test, P0 = 0.012).
FIG. 3.
FIG. 3.
The level of chromosomal fragmentation in the mutT mutants. The seqA mutants are shown as an example of a significant chromosomal fragmentation (44). (A) A representative pulsed-field gel of the chromosomal DNA isolated in agarose plugs from the indicated strains, grown at the indicated temperatures. To the right, the last two lanes after ethidium bromide staining show their relation to molecular weight markers (yeast chromosomes; size indicated in kbp). Strains are the following: wild type, AB1157; recBC(Ts), SK129; ΔmutT, ER2; ΔmutT recBC(Ts), ER3; ΔseqA, ER15; ΔseqA recBC(Ts), ER46. (B) The level of chromosomal fragmentation, averaged from three independent experiments run on different days. The strains are the same as in panel A. The data are means ± standard error of the mean (n = 3). The level of fragmentation in ΔmutT recBC(Ts) at 37°C is not significantly different from that in recBC(Ts) at 37°C (t test, P0 = 0.12). (C) The level of chromosomal fragmentation in the ΔmutT ΔrecBCD mutant, averaged from seven independent experiments run on different days. The data are means ± standard error of the mean. The strains are the following: wild type, AB1157; ΔmutT, ER2; ΔrecBCD, ER8; ΔmutT ΔrecBCD, ER14.
FIG. 4.
FIG. 4.
The level of in vivo plasmid relaxation in a mutT mutant. (A) Total plasmid DNA of pX25A8L (30.0 kbp) was extracted from wild-type (AB1157), ΔmutT mutant (ER2), and dut-1 mutant (AK105) strains as described previously (43) and run on a 1.1% agarose, and the fraction of supercoiled plasmid species in the total plasmid DNA was determined after blot hybridization with plasmid-specific probes. The data points are means ± standard error of the mean (n = 11). The dut value is significantly different from the wild-type value (t test, P0 = 0.025). (B) The procedure described in panel A was performed with plasmids pK96 and RP4. The data points are means ± standard error of the mean (4 ≤ n ≤ 10). The plasmid size is shown in parentheses. The differences in the levels of supercoiling likely reflect the different replicons.
FIG. 5.
FIG. 5.
High copy number of mutM+ and mutY+ does not kill mutT rec mutants. (A) The effect of high-copy-number plasmids carrying the mutM+ or mutY+ genes on the viability of mutT and recBC mutant strains. A total of 10 μl of 10−4 dilutions of rapidly growing cultures was spotted on LB plates and incubated either at 27°C for 36 h or at 42°C for 20 h. Strains are as follows: wild type, AB1157; recBC(Ts), SK129; ΔmutT, ER2; ΔmutT recBC(Ts), ER3. Plasmids are the following: mutM+, pER6; mutY+, pER5. (B) No mutT rec synthetic lethality even when MutM is overexpressed. A total of 10 μl of 10−6 dilutions of saturated cultures was spotted on LB plates either supplemented with IPTG to induce mutM+ expression or left untreated (no induction) and incubated at 30°C for 24 h. The left three spots on each plate are three independent cultures carrying the pTrc99A vector plasmid; the right three spots are three independent cultures carrying the overexpression pMutM plasmid. The strains are the following: wild type, AB1157; ΔmutT, ER2; ΔrecA, JC10287; ΔrecBCD, ER8; ΔmutT ΔrecA, ER5; ΔmutT ΔrecBCD, ER14.
FIG. 6.
FIG. 6.
The level of MutM-recognized DNA modifications in plasmid DNA. (A) RP4 DNA (positive control), treated in vitro with methylene blue (MB) and light, can be subsequently nicked in vitro with the MutM (Fpg) enzyme, indicating the presence of 8-oxo-guanine residues. Amount of MutM is expressed in 102 units, so that, for example, 8 corresponds to 0.08 units. RC, relaxed circular plasmid DNA; SC, supercoiled plasmid DNA; Chr, chromosomal DNA. (B) The level of relaxation caused by MutM-recognized DNA modifications in plasmid DNA isolated from growing cultures of wild-type (AB1157), ΔmutT (ER2), mutM (ER12), and ΔmutT mutM (ER13) strains. Mock, plasmid incubated in the buffer only; treated, plasmid incubated in the complete reaction mixture; difference, the result of the subtraction of the mock value from the treated value. The data points are means of five independent measurements done on different days ± standard error of the mean. (C) The level of relaxation caused by MutM-recognized DNA modifications in plasmid DNA isolated from stationary cultures of the same strains.
FIG. 7.
FIG. 7.
Mutagenesis tests of mutT mutants and their derivatives. (A) Rifampin test. (B) Lac+ reversion in CC101 (a specific test for AT→CG transversion). Values are medians ± first and third quartiles (n = 4 to 19).
FIG. 8.
FIG. 8.
High copy numbers of mutM+ and mutY+, as well as other potential defects in 8-oxo-dGTP interception, do not kill mutT rec mutants. (A) The effect of high-copy-number plasmids carrying the mutM+ or mutY+ genes on the viability of the recA(Ts) mutT ribA triple mutant (ER51). A total of 10 μl of serial dilutions of saturated cultures was spotted on LB plates and incubated at either 30°C or 42°C for 24 h. pMutM+, pER6 plasmid; pMutY+, pER5 plasmid. (B) The recA200(Ts) mutT ribA orf135 quadruple mutant is viable. Ten microliters of serial dilutions of saturated cultures was spotted on LB plates and incubated at either 30°C or 42°C for 24 h. Strains are the following: recA(Ts), JC9941; ΔmutT recA(Ts), ER4; ΔmutT Δorf135 recA(Ts), ER54; ΔmutT Δorf135 ΔribA recA(Ts), ER56.
FIG. 9.
FIG. 9.
Adaptive mutagenesis in CC101 strain. Four independent 12-ml cultures were grown in LB medium from fresh single colonies to saturation and plated on M9 plates supplemented with Lac and incubated for 8 days at 37°C. Each day, the total number of Lac+ colonies on these plates was counted, and the results are shown in this graph. Culture 3 had an initial jackpot but then showed a similar rate of appearance of additional Lac+ colonies as cultures 1 and 4, while culture 2 originally had a normal, low level of Lac+ colonies but then showed an increased adaptive mutation rate relative to the other three cultures.

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