A high level of transcription has been associated with elevated spontaneous mutation and recombination rates in eukaryotic organisms. To determine whether the transcription level is directly correlated with the degree of genomic instability, we have developed a tetracycline-regulated LYS2 reporter system to modulate the transcription level over a broad range in Saccharomyces cerevisiae. We find that spontaneous mutation rate is directly proportional to the transcription level, suggesting that movement of RNA polymerase through the target initiates a mutagenic process(es). Using this system, we also investigated two hypotheses that have been proposed to explain transcription-associated mutagenesis (TAM): 1) transcription impairs replication fork progression in a directional manner and 2) DNA lesions accumulate under high-transcription conditions. The effect of replication fork progression was probed by comparing the mutational rates and spectra in yeast strains with the reporter gene placed in two different orientations near a well-characterized replication origin. The effect of endogenous DNA damage accumulation was investigated by studying TAM in strains defective in nucleotide excision repair or in lesion bypass by the translesion polymerase Polζ. Our results suggest that both replication orientation and endogenous lesion accumulation play significant roles in TAM, particularly in terms of mutation spectra.

High levels of transcription in Saccharomyces cerevisiae are associated with increased genetic instability, which has been linked to DNA damage. Here, we describe a pGAL-CAN1 forward mutation assay for studying transcription-associated mutagenesis (TAM) in yeast. In a wild-type background with no alterations in DNA repair capacity, ≈50% of forward mutations that arise in the CAN1 gene under high-transcription conditions are deletions of 2-5 bp. Furthermore, the deletions characteristic of TAM localize to discrete hotspots that coincide with 2-4 copies of a tandem repeat. Although the signature deletions of TAM are not affected by the loss of error-free or error-prone lesion bypass pathways, they are completely eliminated by deletion of the TOP1 gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed into the context of a frameshift reversion assay, which is sensitive enough to detect Top1-dependent deletions even in the absence of high transcription. We suggest that the accumulation of Top1 cleavage complexes is related to the level of transcription and that their removal leads to the signature deletions. Given the high degree of conservation between DNA metabolic processes, the links established here among transcription, Top1, and mutagenesis are likely to extend beyond the yeast system.

High levels of transcription are associated with increased mutation rates in Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation (TAM). To obtain insight into the mechanism of TAM, we obtained LYS2 forward mutation spectra under low-versus high-transcription conditions in which LYS2 was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10 promoter, respectively. Because of the large size of the LYS2 locus, forward mutations first were mapped to specific LYS2 subregions, and then those mutations that occurred within a defined 736-bp target region were sequenced. In the low-transcription strain base substitutions comprised the majority (64%) of mutations, whereas short insertion-deletion mutations predominated (56%) in the high-transcription strain. Most notably, deletions of 2 nucleotides (nt) comprised 21% of the mutations in the high-transcription strain, and these events occurred predominantly at 5 -(G/C)AAA-3 sites. No ؊2 events were present in the low-transcription spectrum, thus identifying 2-nt deletions as a unique mutational signature for TAM.Transcription influences genomic stability in a complex manner by affecting DNA repair, recombination, mutagenesis, and chromatin structure (reviewed in references 1 and 31). In the subpathway of nucleotide excision repair known as transcription-coupled repair, for example, the encounter of RNA polymerase (Pol) with a transcription-blocking lesion on the transcribed strand specifically triggers repair of the damage, resulting in more efficient repair of lesions on the transcribed strand than on the nontranscribed strand (16). In addition to promoting strand-specific repair, high levels of transcription have been shown to elevate recombination rates in Saccharomyces cerevisiae (33,36,39,41), Schizosaccharomyces pombe (15), and mammalian cells (34). Finally, increased transcription has been shown to stimulate spontaneous mutagenesis in Escherichia coli (3,24,44) and bacteriophage T7 (4) and to enhance deletions within an E. coli plasmid target (40). In S. cerevisiae, Datta and Jinks-Robertson demonstrated previously that an increased transcription level likewise stimulates spontaneous mutation rates, a phenomenon termed transcriptionassociated mutation, or TAM (8). Specifically, reversion of the lys2⌬Bgl frameshift allele and forward mutation at the LYS2 locus increased 35-and 10-fold, respectively, in response to transcriptional induction from the pGAL1-10 promoter. Whereas the genetic requirements and underlying mechanisms of transcription-coupled repair (16, 38) and transcription-associated recombination are becoming clearer (11,12,21), the mechanism of TAM is still poorly understood.The genetic requirements and mutation spectrum of TAM have been characterized in yeast strains using lys2⌬Bgl, a ϩ4 frameshift allele that reverts by acquisition of compensatory Ϫ1 frameshift mutations. In the high-transcription strain, 70% of the reversion mutations required Rev3p, a component of the translesion synthesis DNA Pol , sugg...

To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (Mf), and glycophorin A (GPA) variant erythrocytes (Vf) of both allele-loss (phi/N) and allele-loss-and-duplication (N/N) phenotypes in umbilical cord blood. The mean hprt Mf (1.40 +/- 1.11 x 10(-6), N = 66) and GPA Vf (phi/N 4.0 +/- 2.2 x 10(-6), N = 114; N/N 2.7 +/- 2.0 x 10(-6), N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt Mf was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 +/- 0.41 x 10(-6), N = 45, P < 0.01; t test, P < 0.01, Mann-Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt Mf (0.70 +/- 0.49 x 10(-6)) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann-Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated Mf revealed a significant decrease in the relative contribution of gross structural mutations to the overall Mf (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, chi 2 test), suggesting that the higher Mf resulted from an elevated level of "point" mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt Mf or GPA Vf.

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