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. 1998 Oct 13;95(21):12468-73.
doi: 10.1073/pnas.95.21.12468.

Induction of microsatellite instability by oxidative DNA damage

A L Jackson  1 , R Chen, L A Loeb
Affiliations

Induction of microsatellite instability by oxidative DNA damage

A L Jackson et al. Proc Natl Acad Sci U S A. .

Abstract

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the beta-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of beta-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the beta-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 10(6) wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency approximately 300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N'-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This beta-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

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Figures

Figure 1
Figure 1
Assay for the selection of oxidative damage-induced frameshift mutations in repetitive sequences. A modified version of the low copy number plasmid pBR322 was constructed to contain the tetracycline resistance gene and a carbenicillin resistance gene containing an out-of-frame microsatellite sequence. This construct renders bacteria tetracycline-resistant and carbenicillin-sensitive. Following treatment with H2O2, damaged plasmid was transformed into wild-type BL21 E. coli. Colony growth on tetracycline-containing plates was indicative of plasmid survival, whereas growth on tetracycline plus carbenicillin was indicative of a compensating frameshift mutation to restore the reading frame to generate functional β-lactamase. Mutation frequency is calculated as the number of carbenicillin-resistant mutants recovered per tetracycline-resistant survivor.
Figure 2
Figure 2
Induction of dinucleotide frameshift mutations by hydrogen peroxide. Plasmid DNA (9 × 10−15 mol) containing either repetitive sequences or control nonrepetitive sequences within the β-lactamase gene was exposed to 1 μM or 50 μM H2O2. The 1 μM H2O2 treatment corresponded to a 40% survival of plasmid relative to untreated plasmid as measured by tetracycline-resistant transformants, whereas 50 μM H2O2 treatment corresponded to 20% survival. Dinucleotide (CA)11 or (CA)12 repeats and control nonrepetitive polylinker in +2 frame are shown.
Figure 3
Figure 3
Induction of mononucleotide frameshift mutations by hydrogen peroxide. Treatment conditions were identical to those for dinucleotide repeats in Fig. 2. Mononucleotide (A)10 or (G)10 repeats and control nonrepetitive polylinker in +1 frame are shown.
Figure 4
Figure 4
Modulation of frameshift frequency by exposure to oxygen radicals. Plasmid containing a +2 frame (CA)11 microsatellite sequence within the β-lactamase gene was exposed to 1 μM H2O2 or 50 μM H2O2 for 1 h at room temperature in the presence of either 1 unit catalase, 0.5 μM CuCl2, or 0.5 μM NiCl2. Addition of 0.5 μM Cu2+ or Ni2+ in the presence of 1 μM H2O2 reduced the recovery of tetracycline-resistant transformants to 30% relative to H2O2 treatment alone. In the presence of 50 μM H2O2 and 0.5 μM Cu2+, no survivors were detected.
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