Roles of Saccharomyces cerevisiae DNA polymerases Poleta and Polzeta in response to irradiation by simulated sunlight

Abstract

Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans. Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase eta (Poleta) and polymerase zeta (Polzeta), encoded by the RAD30A and REV3 genes, respectively. Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm). However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310-1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions. Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Delta, rev3Delta and rev3Delta rad30Delta strains. The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6-4) photoproducts derived from studies with UVC. They further suggest that Poleta participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polzeta is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts. They also suggest that in the absence of Polzeta, Poleta contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine.

Figure 1

Sensitivity of wild-type (circles), rad30 (triangles) and rev3 (diamonds) strains to SSL (A) and UVC (B), and induction of can1 mutants in the wild-type (circles) and rad30 (triangles) strains by SSL (C) and UVC (D). The data from each of at least three independent experiments are represented as open symbols and the means are represented as closed symbols. Values for these means are shown near each closed symbol. The differences observed between wild-type and other strains in all cases were statistically significant (linear multivariate regression analysis, P < 0.01).

Figure 2

Mutation frequencies as a function of survival level. Frequencies of can1 mutants in the wild-type (circles) and rad30 (triangles) strains upon irradiation with SSL (closed symbols) or UVC (open symbols) were plotted against corresponding surviving fractions.

Figure 3

Distribution of UVC- and SSL-induced mutations along the CAN1 gene. Mutations that occurred in the wild-type (blue), rad30 (red), rev3 (green) and rev3 rad30 (black) strains are shown. The sequence is that of the non-transcribed strand. Only 1 nt alterations are shown. SSL- and UVC-induced changes are represented above and below the sequence, respectively. Tandem and non-tandem closely spaced double mutations are boxed. The lower case letters in the gene sequence indicate the altered base. Altered bases in the gene sequence where mutations occurred in more than one strain are underlined.

Figure 4

The frequencies and types of base changes associated with defined sites induced by SSL (A and B) and UVC (C and D). Changes indicated: T→A (open bars), T→G (horizontal lined bars), T→C (vertical lined bars), C→T (oblique bars), C→A (black bars), C→G (dotted bars). Overlapped sites (CCT, TTC, TCC and CTT) are indicated as OVL and non-bipyrimidine sites as NBP.

Figure 5

Position specificity (3′ or 5′) of base substitutions at bipyrimidine sites 5′CC3′, 5′TT3′, 5′TC3′ and 5′CT3′ induced by SSL (above the sites) and UVC (below the sites). Tandem double mutations are boxed.