Mutation frequency analysis of mononucleotide and dinucleotide repeats after oxidative stress

Abstract

Many tumors exhibit genetic instability at the DNA sequence level in the form of frameshift mutations in simple repeats (microsatellite instability). A high level of microsatellite instability, such as that seen in hereditary nonpolyposis colorectal cancer (HNPCC), arises from defects in the mismatch repair pathway. A low level of microsatellite instability is found in some non-HNPCC-associated cancers, such as those of the breast and lung, and is not attributable to mismatch repair defects. We hypothesized that oxidative DNA damage may be at least partly responsible for the generation of microsatellite mutations in these tumors. We investigated whether oxidative DNA damage can induce microsatellite mutations in mismatch repair-proficient cultured cells. Telomerase-immortalized normal human fibroblasts were stably transfected with a plasmid containing a tk-neo fusion gene, such that the neo coding region was placed out of frame by the presence of an upstream microsatellite sequence. Cells were treated with H(2)O(2) and mutation frequencies were determined for G(17), A(17), and (CA)(17) repeats. Mutation frequencies of mononucleotide repeats in cells with the neo gene in the (+1) reading frame were reduced after treatment. No effect was observed in cells with the mononucleotide repeats in the (-1) reading frame. A small increase in mutation frequency was observed in cells with the (CA)(17) repeat. Our data suggest that diploid human cells may have protective mechanisms that prevent the induction of microsatellite mutations by a short exposure to high levels of oxidative stress.

A genome-wide view of the spectrum of spontaneous mutations in yeast

“…1), consistent with previous observations from C. elegans, which indicate an ϳ140-fold inflation of the mutation rate for mononucleotide runs of 12 bp relative to loci containing the same number of dinucleotide repeats (7,23). Analyses with human cell lines also suggest much greater instabilities for mononucleotide than dinucleotide repeats (30). Nevertheless, we emphasize that a precise determination of the mutational profile for nuclear HPR loci in yeast remains to be made.…”

Models for chromosomal replication‐independent non‐B DNA structure‐induced genetic instability

“…However, H 2 O 2 treatment on telomerase‐immortalized normal human fibroblasts harboring a plasmid containing G(17), A(17), or CA(17) repeats in the tk‐neo gene, reduced mononucleotide repeat slippage‐induced reactivation of the reporter neo gene in the (+1) reading frame. No effect was observed when the neo gene was in the (−1) reading frame 75. These observations suggested that the chemical and/or structural nature of different types of repetitive DNA might be important for the fate of the damaged sequence.…”

Tumor hypoxia and genetic alterations in sporadic cancers

“…They speculated that ROS induces low levels of mutations in di-nucleotide repeats. 51 In accordance with the effect of ROS on microsatellite loci in human cells, Chang et al reported that non-toxic levels of H2O2 impair mismatch repair activity, 52 which leads to DNA slippage mutations at microsatellite loci (see below).…”

“…They found that H 2 O 2 treatment decreased the mutation frequency of mononucleotide repeats, but increased the mutation frequency of di‐nucleotide repeats in non‐cancer diploid human cells. They speculated that ROS induces low levels of mutations in di‐nucleotide repeats 51 . In accordance with the effect of ROS on microsatellite loci in human cells, Chang et al .…”