The mechanisms by which imbalanced dNTPs induce mutations have been well characterized within a test tube, but not in vivo. We have examined mechanisms by which dNTP imbalances induce genome instability in strains of Saccharomyces cerevisiae with different amino acid substitutions in Rnr1, the large subunit of ribonucleotide reductase. These strains have different dNTP imbalances that correlate with elevated CAN1 mutation rates, with both substitution and insertion–deletion rates increasing by 10- to 300-fold. The locations of the mutations in a strain with elevated dTTP and dCTP are completely different from those in a strain with elevated dATP and dGTP. Thus, imbalanced dNTPs reduce genome stability in a manner that is highly dependent on the nature and degree of the imbalance. Mutagenesis is enhanced despite the availability of proofreading and mismatch repair. The mutations can be explained by imbalanced dNTP-induced increases in misinsertion, strand misalignment and mismatch extension at the expense of proofreading. This implies that the relative dNTP concentrations measured in extracts are truly available to a replication fork in vivo. An interesting mutational strand bias is observed in one rnr1 strain, suggesting that the S-phase checkpoint selectively prevents replication errors during leading strand replication.

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.

UV irradiation, a known carcinogen, induces the formation of dipyrimidine dimers with the predominant lesions being cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone adducts (6-4PPs). The relative roles of the yeast translesion synthesis DNA polymerases Polz and Polh in UV survival and mutagenesis were examined using strains deficient in one or both polymerases. In addition, photoreactivation was used to specifically remove CPDs, thus allowing an estimate to be made of the relative contributions of CPDs vs. 6-4PPs to overall survival and mutagenesis. In terms of UV-induced mutagenesis, we focused on the 11 frameshift mutations detected by reversion of the lys2DA746 allele, as Polz produces a distinct mutational signature in this assay. Results suggest that CPDs are responsible for most of the UVassociated toxicity as well as for the majority of UV-induced frameshift mutations in yeast. Although the presence of Polh generally suppresses UV-induced mutagenesis, our data suggest a role for this polymerase in generating some classes of 11 frameshifts. Finally, the examination of frameshift reversion spectra indicates a hierarchy between Polh and Polz with respect to the bypass of UV-induced lesions. U LTRAVIOLET (UV) radiation derived from sunlight is a potent mutagen associated with the development of skin cancer in humans. Long-wave UVA (320-400 nm) damages DNA indirectly via the production of reactive oxygen species (Kozmin et al. 2005), while UVB and UVC (290-320 nm and 100-290 nm, respectively) directly induce the formation of covalent linkages between adjacent pyrimidines (Ravanat et al. 2001). The two major forms of such linkages are the abundant cyclobutane pyrimidine dimers (CPDs) and the less abundant pyrimidine (6-4) pyrimidone adducts (6-4PPs) (reviewed in Taylor 2006). The highly conserved nucleotide excision repair (NER) pathway (for a review, see Prakash and Prakash 2000) is the predominant pathway for repairing UV-induced pyrimidine dimers, and organisms that lack this pathway exhibit enhanced sensitivity to the lethal and mutagenic effects of UV (Friedberg et al. 1995). In humans, a lack of NER is associated with the disease xeroderma pigmentosum (XP), which is characterized by an exquisite sensitivity to UV light and an extraordinarily high incidence of skin cancer (Hoeijmakers 2001). In addition to the NER pathway, some organisms, including Saccharomyces cerevisiae, have the ability to directly reverse CPDs enzymatically in a process called photoreactivation (PR). In PR, a DNA photolyase binds to a CPD and utilizes the energy associated with 365-385 nm light to cleave the cyclobutane ring and regenerate the two adjacent pyrimidine bases (reviewed in Sancar 2006). Because 6-4PPs cannot be similarly reversed in yeast, the relative effects of CPDs and 6-4PPs on survival and mutagenesis can be deduced by measuring these biological endpoints in the presence vs. absence of PR.UV-induced lesions cannot be bypassed by the replicative DNA polymerases and, if not removed by NER o...

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