The in Vivo Characterization of Translesion Synthesis Across UV-Induced Lesions in Saccharomyces cerevisiae: Insights Into Polζ- and Polη-Dependent Frameshift Mutagenesis

Abstract

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...

Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance

“…The phenotypes of mutants with DNA Pol deficiencies and the in vitro activity of Pol indicate that its major role is the nonmutagenic bypass of UV-induced DNA lesions. In particular, Pol is the primary TLS polymerase responsible in many organisms for error-free bypass of cis-syn cyclobutane pyrimidine dimers (CPDs), one of the major lesions resulting from UV radiation (1,63,263). In vitro, Pol has been shown to bypass CPDs with high accuracy and efficiency (100), and in vivo, it is thought to be responsible for restarting stalled replication forks and allowing continuous DNA synthesis past sites of UV damage (132).…”

A Polymerase – Tautomeric Model for Targeted Frameshift Mutations: Deletions Formation during Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers

“…The most common lesions are cis-syn cyclobutane pyrimidine dimers [4], in which the bases preserve their original orientation relative to the sugarphosphate backbone [7]. Cyclobutane pyrimidine dimers and (6-4) adducts cause substutution mutations [8][9][10][11][12][13][14][15][16][17][18][19], frameshifts [11,20,21], and complex mutations [22]. Only a few photodimers result in mutations, while more than 90% of these lesions do not [9].…”

Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations

“…Polζ-deficient strains show higher sensitivity to low doses of UV light than Polη mutants, while Polη-deficient strains are more sensitive to higher doses (>30 J/m 2 ; (70)). These data imply that the bypass of UV-induced lesions at lower doses relies predominantly on Polζ, while other polymerases become important at higher doses.…”