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 Pol and Pol 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 +1 frameshift mutations detected by reversion of the lys2A746 allele, as Pol produces a distinct mutational signature in this assay. Results suggest that CPDs are responsible for most of the UV-associated toxicity as well as for the majority of UV-induced frameshift mutations in yeast. Although the presence of Pol generally suppresses UV-induced mutagenesis, our data suggest a role for this polymerase in generating some classes of +1 frameshifts. Finally, the examination of frameshift reversion spectra indicates a hierarchy between Pol and Pol with respect to the bypass of UV-induced lesions.

---

ULTRAVIOLET (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 or reversed by photolyase, have the potential to block the progress of the replication fork. Such replication-blocking lesions can be dealt with by one of two general tolerance/bypass pathways: template switching or translesion synthesis (TLS) (BARBOUR and XIAO 2003). In template switching the lesion-blocked primer strand uses the complementary strand of the sister chromatid as a template to bypass the damage. This can either involve annealing between the nascent leading and lagging strands at the replication fork or occur by homologous recombination with the duplex sister being invaded by the blocked nascent strand. As an alternative to template switching, TLS uses specialized DNA polymerases with generally low fidelity and low processivity to insert and/or extend nucleotides across from a lesion (RATTRAY and STRATHERN 2003). This pathway allows the cell to bypass lesions and continue replication, but often at the expense of increased mutagenesis.

S. cerevisiae has three TLS polymerases: Pol, Rev1, and Pol (RATTRAY and STRATHERN 2003). Pol, a complex of the Rev3 and Rev7 proteins, is required for >90% of induced mutagenesis and hence has been designated as an error-prone polymerase (LAWRENCE 2002). In vitro, purified Pol bypasses lesions such as CPDs in a highly mutagenic manner (NELSON et al. 1996), but bypasses other lesions such as thymine glycols in a relatively error-free manner (JOHNSON et al. 2003). Pol appears to be most efficient, however, at extending from a nucleotide inserted across from a lesion or extending mismatched termini (PRAKASH and PRAKASH 2002). This unique extension activity has led to the view that Pol may work primarily in conjunction with other DNA polymerases in lesion bypass (PRAKASH and PRAKASH 2002). With regard to the bypass of CPDs vs. 6-4PPs in vivo, a transformation-based gap-filling assay has indicated a central role for Pol in bypassing 6-4PPs, but a relatively minimal role in bypassing CPDs (GIBBS et al. 2005). Rev1, a member of the Y family of DNA polymerases, is generally thought to be required for in vivo Pol activity, although there have been sporadic reports suggesting that there may be exceptions to this generality (BAYNTON et al. 1999). In addition, Rev1 has an independent deoxycytidyl transferase activity, specifically incorporating dCMP opposite abasic sites in vitro (NELSON et al. 1996; HARACSKA et al. 2002).

Pol is encoded by the RAD30 gene and, like Rev1, is a member of the Y family of DNA polymerases (MCDONALD et al. 1997; ROUSH et al. 1998). Pol was originally characterized on the basis of its unique ability to replicate past thymine–thymine dimers with high fidelity in vitro (JOHNSON et al. 1999), and yeast studies support a role for Pol in the error-free bypass of CPDs. Pol's role in the bypass of 6-4PPs is more controversial with some studies suggesting a predominant role for Pol in the bypass of 6-4PPs (BRESSON and FUCHS 2002) and others suggesting only a minor role for this TLS polymerase (GIBBS et al. 2005). A lack of Pol in humans is associated with a variant form of XP in which the UV damage normally bypassed in an error-free manner by hPol is presumably bypassed by a much more mutagenic translesion polymerase (KANNOUCHE and STARY 2003). In spite of its original designation as an error-free TLS polymerase, Pol has very low fidelity when copying undamaged DNA templates (WASHINGTON et al. 1999) and is important for UV-induced mutagenesis in at least some yeast assays (ZHANG and SIEDE 2002).

Most studies of UV-induced mutagenesis in yeast have focused on base substitutions in either forward or reverse mutation assays (KUNZ et al. 1987; MCDONALD et al. 1997; YU et al. 2001; ZHANG and SIEDE 2002; KOZMIN et al. 2003). In forward mutation spectra of the CAN1 and SUP4-o genes, for example, >95% of the UV-induced events were base substitutions (ARMSTRONG and KUNZ 1990; KOZMIN et al. 2003). One yeast study that compared induced reversion frequencies of cyc1 missense vs. frameshift alleles concluded, however, that UV-induced frameshifts may constitute 10–20% of the total mutations (LAWRENCE et al. 1984). In the current study, a reversion assay is used to focus specifically on the production of UV-induced frameshift mutations. These experiments employ the chromosomal lys2A746 frameshift allele, which reverts by acquisition of a compensatory +1 frameshift mutation within a defined 150-bp reversion window (HARFE and JINKS-ROBERTSON 1999). Because Pol produces a unique mutational signature in this assay system, appropriate mutant strains can be used to deduce the relative roles of Pol and Pol in UV-induced frameshift mutagenesis. In addition, survival and mutagenesis were assessed both in the presence and in the absence of photoreactivation, thereby allowing the relative contributions of CPDs and 6-4PPs to be determined. These studies reveal dose-dependent differences in survival and mutagenesis and provide further evidence that Pol and Pol play partially redundant roles in the bypass of CPDs and 6-4PPs.

Media and growth conditions:

Yeast strains were grown nonselectively in YEP medium (1% yeast extract, 2% Bacto-peptone, 250 mg/liter adenine, 2% agar for plates), which was supplemented with 2% dextrose (YEPD). Selective growth was on synthetic complete medium containing 2% dextrose (SCD) and lacking the appropriate nutrient (SHERMAN 1991). Canavanine-resistant mutants in the forward mutation assay were identified on SCD–Arg plates supplemented with 60 µg/ml canavanine. All growth was at 30°.

Strain construction:

Mutant strains were derived from SJR922 (MAT ade2-101_oc his3Nco lys2A746; HARFE and JINKS-ROBERTSON 1999) by lithium acetate transformation (GIETZ and WOODS 2002). The rev3 strain, SJR1272, was constructed using a PCR-generated rev3::kan fragment (SWANSON et al. 1999). SJR1463 contains the rad30::HIS3 allele and was constructed by transformation with SpeI-digested pJM82 (MCDONALD et al. 1997). The rev3rad30 double mutant was created by introducing rev3::kan into SJR1463.

UV survival and mutagenesis:

Cultures inoculated from single colonies were grown nonselectively in 5 ml YEPD to saturation (2 x 10⁸ cells/ml). Cells were washed with H₂O, resuspended in 1–1.5 ml of H₂O, and 100 µl of the appropriate cell dilution were plated on YEPD and the appropriate SCD-based selective media to assess cell survival and mutagenesis, respectively. YEPD was used to assess UV-induced cell killing, with the survival data being indistinguishable from those obtained when using SCD medium. Within 1 hr of plating, cells were exposed to the specified dose of 254 nm UV light using a UV crosslinker (Ultra Lum). Immediately after UV exposure the plates were wrapped in aluminum foil to prevent photoreactivation. For photoreactivation experiments, plates were exposed to 365 nm of UVA light for 20 min (11 J/m²/sec), using a long-wave UV transilluminator (Spectroline). Because longer exposures to UVA (up to 60 min) did not improve survival, we assume that all of the CPDs that were capable of being photoreversed had been removed within the 20 min used in our experiments. Colonies arising on YEPD and SCD plates were counted after 2 and 3 days of incubation, respectively. Each data point corresponds to the mean of four to six independent survival/mutagenesis experiments, and error bars represent the standard deviation.

lys2A746 reversion spectra:

Genomic DNA was isolated from purified Lys⁺ colonies using a glass bead lysis procedure. A portion of the LYS2 locus containing the reversion window was amplified by PCR and DNA sequence analysis of PCR-amplified genomic fragments was performed by Macrogen (Seoul, South Korea), using the primer 5'-CGCAACAATGGTTACTCT. Sequences were analyzed using the Sequence Manager software (DNASTAR, Madison, WI) licensed from BIMCORE at Emory University.

The relative roles of the yeast TLS polymerases Pol and Pol in survival and mutagenesis following UVC irradiation were determined using a wild-type (WT) strain, a strain lacking Pol (rev3 mutant), a strain lacking Pol (rad30 mutant), and a strain simultaneously lacking both Pol and Pol (rev3 rad30 double mutant). Because rev1 and rev3 mutants typically exhibit indistinguishable UV-induced toxicity/mutagenesis and Rev1 and Pol are generally assumed to act in concert to bypass UV-induced lesions (LAWRENCE 2002), we did not examine rev1 mutants in our studies. Cells were grown to saturation in nonselective liquid medium, plated selectively or nonselectively as appropriate, and then irradiated with varying doses of UV up to 80 J/m². Two different assays were used to assess mutagenesis. First, the forward mutation frequency at the CAN1 locus was measured to assay a very broad range of mutation types. Second, the level of frameshift mutagenesis was assessed using the lys2A746 allele, which specifically detects net +1 frameshift mutations (HARFE and JINKS-ROBERTSON 1999). This particular allele was used because of the very distinctive signature attributable to Pol in the corresponding reversion spectra, thus allowing a detailed assessment of Pol-dependent mutagenesis. Specifically, Pol is required for the production of "complex" reversion events, which are defined as frameshift mutations accompanied by one or more closely spaced base substitutions (HARFE and JINKS-ROBERTSON 2000; MINESINGER and JINKS-ROBERTSON 2005). Finally, the relative contributions of the two major classes of UV damage, CPDs and 6-4 PPs, to UV-associated killing and mutagenesis were estimated following the specific reversal of CPDs by PR.

Survival following UV irradiation:

Some studies have reported that a Pol-deficient, rev3 mutant is more sensitive to UV light than is a Pol-deficient, rad30 mutant (KOZMIN et al. 2003), while others concluded the reverse (MCDONALD et al. 1997; YU et al. 2001). These results can be reconciled by the survival curves shown in Figure 1A, where there is a clear reversal in the relative sensitivities of the rev3 and rad30 strains at a UV dose of 40 J/m². The rev3 strain was thus more sensitive to UV irradiation than was a rad30 strain at low UV doses, while the relative sensitivities of the mutants were reversed at the higher UV doses (see also MCDONALD et al. 1997). We suggest that the greater survival-promoting role for Pol at higher UV doses may partially reflect the documented UV induction of RAD30 transcription (MCDONALD et al. 1997; ROUSH et al. 1998). Either a dose-dependent induction or the requirement for a threshold level of UV damage for RAD30 induction could, in principle, account for the greater dependence on Pol for tolerating lesions produced by low UV doses.

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 1.— Survival as a function of UVC dose in the (A) absence or (B) presence of photoreactivation (PR). WT (•, ), rev3 (, ), rad30 (, ), and rev3 rad30 (x) strains are shown. Error bars correspond to the standard deviation. (C and D) The relative toxicities of CPDs (open symbols) vs. 6-4PPs (shaded symbols) in the rev3 and rad30 mutants, respectively. The toxicity associated with CPDs at a given UV dose was calculated by dividing the UV – PR survival at that dose by UV + PR survival.

 
As reported previously (MCDONALD et al. 1997; YU et al. 2001), the rev3 rad30 double mutant was much more sensitive than either single mutant to UV doses exceeding 30 J/m², suggesting functionally redundant roles for Pol and Pol in the bypass of a common UV lesion. In contrast to the synergism between rev3 and rad30 observed at high UV doses, however, at the lowest doses where the rad30 mutant had begun to exhibit clear UV sensitivity (10–20 J/m²), the rev3 rad30 double mutant was no more sensitive than was the rev3 single mutant. The epistasis of rev3 to rad30 at low UV doses suggests that Pol and Pol may act together to bypass some types of UV-induced lesions, and that Pol additionally can promote UV survival in a manner that is independent of Pol.

The survival curves when UV exposure was followed by 20 min of photoreactivation (UV + PR) are shown in Figure 1B. Photoreactivation in the absence of prior UV irradiation had no effect on survival, and even at the highest UV dose used, the maximum recovery of viability was obtained within the 20-min period of PR (data not shown). Although we assume that PR removes the majority of, if not all, CPDs, some protection from removal may be provided by positioned, static nucleosomes (THOMA 1999). For the sake of discussion, it is assumed that lesions remaining after PR are composed predominantly of 6-4PPs, although other minor photoproducts (TAYLOR 2006) as well as residual CPDs may be present. The most notable feature of the UV + PR data is the coincidence of the rev3 and rev3 rad30 survival curves, with these strains being more sensitive to 6-4PPs than the WT and rad30 strains. These data suggest a requirement for Pol in bypassing 6-4PPs, with little, if any, ability of Pol alone to bypass this lesion. These data are consistent with the demonstrated role of Pol, but not of Pol, in 6-4PP bypass in in vivo gap repair assays (GIBBS et al. 2005) and contradict the idea that Pol might play a major, Pol-independent role in the global bypass of 6-4PPs (BRESSON and FUCHS 2002). If Pol requires another polymerase for 6-4PP bypass, this role would presumably be filled by Rev1 or by one of the replicative DNA polymerases.

The effect of 6-4PPs on survival is reflected by the percentage of survival following the removal of CPDs by PR (Figure 1B, UV + PR). The effect of CPDs on survival at each UV dose can be similarly estimated as the ratio of the survival before PR to that after PR [(UV – PR)/(UV + PR)]. Although this type of calculation makes the simplifying assumption that the effects of UV-induced lesions are completely independent of each other, a partitioning of the UV-killing effects into those attributable primarily to CPDs vs. 6-4PPs is nevertheless informative. The dose-dependent survival profiles of the rev3 and rad30 single-mutant strains without photoreactivity (CPDs + 6-4PPs) and with photoreactivity (6-4PPs), as well as those obtained by taking the ratio of these two measured survival values (CPDs), are presented in Figure 1, C and D. In the rev3 mutant, where Pol is the only functional TLS polymerase, PR had little effect on survival, with 6-4PPs thus being more toxic than CPDs. In contrast, the rad30 mutant, which has Pol as its only TLS polymerase, generally exhibited greater toxicity to CPDs than to 6-4PPs. These data are consistent with the ability of purified Pol to bypass CPDs in vitro (JOHNSON et al. 2001) and with the requirement for Pol in CPD bypass in in vivo gap-filling assays (GIBBS et al. 2005). These data also lend support to in vivo evidence suggesting that the bypass of most 6-4PPs requires Pol activity (GIBBS et al. 2005).

UV-induced mutagenesis without photoreactivation:

In Escherichia coli the majority of UV-induced mutations are base substitutions at dipyrimidine sites, with frameshifts typically composing <25% of the spectra (e.g., see MILLER 1985; SCHAAPER et al. 1987). One of the very striking aspects of UV-induced frameshift mutagenesis in bacteria is that –1 events outnumber +1 events at least 10:1. In yeast, forward mutation studies have been done using either the SUP4-o ochre-suppressing tRNA gene (KUNZ et al. 1987) or the CAN1 locus (KOZMIN et al. 2003) as a target. As in bacterial cells, base substitutions predominate and occur mainly at dipyrimidine sites. Although UV-induced frameshift mutations appear to be proportionally less frequent in yeast than in bacterial cells, a similar strong bias for –1 as opposed to +1 events has been reported. In the current study, we have chosen to focus on UV-induced +1 frameshifts in yeast, primarily because of the unique mutational signature produced by Pol in the lys2A746 reversion assay (HARFE and JINKS-ROBERTSON 2000). To allow comparison to other studies and to provide a context for the UV-induced frameshift mutagenesis, we also examined forward mutation at the CAN1 locus.

A dose-dependent increase in the frequency of UV-induced can1 mutants was observed in the WT background, with an 200-fold increase in can1 mutant frequency at the highest UV dose relative to the spontaneous (no UV) frequency (Figure 2A). Consistent with a role for Pol in the error-free bypass of UV damage, and hence the avoidance of UV-induced mutagenesis, the frequency of UV-induced can1 mutations was consistently elevated in a rad30 mutant relative to the WT strain. As expected, UV-induced mutagenesis was almost completely dependent on Pol activity, as there was little induction of can1 mutants in the rev3 background. There was, however, an unexpected induction of can1 mutants at the highest UV doses in the rev3 rad30 double mutant. In the absence of Pol, there thus appears to be a Pol-independent mechanism for generating UV-induced mutations that eliminate CAN1 activity. We considered the possibility that the corresponding mutants might harbor a complete loss of the CAN1 locus (CHEN and KOLODNER 1999), but because we were still able to PCR amplify the locus in the mutants examined (data not shown), this is unlikely. Whether the Pol-independent can1 mutants reflect a novel mechanism of UV-induced mutagenesis, or might simply be a manifestation of the very low survival of the rev3 rad30 mutant at high UV doses, is not known.

View larger version (20K): In this window In a new window Download PPT slide   FIGURE 2.— UV-induced mutagenesis. Frequencies of UV-induced forward mutation at CAN1 (A) and lys2A746 reversion (B) in WT (•), rev3 (), rad30 (), and rev3 rad30 (x) strains. UV-induced mutation frequencies were calculated by subtracting the frequency in the starting culture (0 J/m2) from that obtained after each specified UV dose. Error bars correspond to the standard deviation.

 
A stronger induction of Lys⁺ revertants than of can1 mutants was evident following UV irradiation; at a dose of 60 J/m², the reversion frequency of the lys2A746 allele was stimulated 800-fold (Figure 2B). The strong UV induction of frameshifts was not unique to the +1 events detected by the lys2A746 reversion assay; a similar level of induction was observed when reversion of the lys2Bgl allele, which reverts via compensatory –1 frameshifts (GREENE and JINKS-ROBERTSON 1997), was examined (data not shown). Thus, in striking contrast to the situation in bacterial cells where –1 frameshifts greatly outnumber +1 frameshifts (MILLER 1985; SCHAAPER et al. 1987), +1 and –1 frameshifts are induced at similar levels at the yeast LYS2 locus. Given the reported rarity of UV-induced frameshifts in yeast forward mutation assays, the slightly greater induction of Lys⁺ revertants than of can1 mutants was surprising. In the case of UV-induced mutations in SUP4-o, the rarity of frameshifts could be related to the nature of the target. The SUP4-o locus is quite small (89 bp) and contains no A/T runs >3 nt (KUNZ et al. 1987), which is where most frameshifts accumulate in the lys2A746 assay (see spectra below). In the case of the UV-induced can1 mutants, only 32 mutants were sequenced and these were isolated following a single UV dose that resulted in only 10–15% killing (KOZMIN et al. 2003). We estimate that at a comparable level of killing in our WT strain (10 J/m² of UV), the frequency of UV-induced Lys⁺ revertants likely would have been 5- to 10-fold lower than that of can1 mutants. On the basis of our analyses, however, we conclude that, as in bacterial cells, frameshift mutations can compose a sizeable fraction of UV-induced mutations in yeast.

As was seen in the CAN1 forward mutation assay, reversion of the lys2A746 allele was elevated slightly in the rad30 background and was reduced in the rev3 background relative to the WT background. In contrast to the near complete dependency of can1 mutations on Pol (PAULOVICH et al. 1998; FRIEDL et al. 2001; STELTER and ULRICH 2003), however, a significant induction of Lys⁺ revertants was detected in the rev3 mutant. These data are consistent with the earlier report that a small proportion of UV-induced his4 frameshifts occur independently of REV3 and REV1 (LAWRENCE et al. 1984). As observed in the CAN1 assay, there was an inexplicable induction of Lys⁺ revertants at high UV doses in the rev3 rad30 double mutant. This strong induction of Lys⁺ revertants also occurred in a rev3 rad30 rev1 triple mutant at high UV doses (data not shown), indicating that the residual frameshift mutagenesis is independent of all three yeast TLS polymerases.

UV-induced mutagenesis with photoreactivation:

As with survival, UV-induced reversion of the lys2A746 allele was examined in the presence vs. absence of PR. At a given UV dose, +1 frameshifts due to the combined action of CPDs and 6-4PPs are reflected by the Lys⁺ frequency in the absence of PR (Figure 3A), while mutagenesis due to 6-4PPs corresponds to the Lys⁺ frequency following the removal of CPDs by PR (Figure 3B). For the WT, rev3, rad30, and rev3 rad30 strains, a comparison of the frequency of Lys⁺ revertants before and after PR indicates that the majority of the UV-induced +1 frameshifts in each strain were due to CPDs. A similar conclusion concerning the relative contributions of CPDs and 6-4PPs to mutagenesis was reached in studies using the SUP4-o forward mutation assay (ARMSTRONG and KUNZ 1992) and in mammalian cells (JANS et al. 2005). Loss of Pol was associated with an increase in the UV-induced lys2A746 reversion frequency in the absence of PR, but its loss had no effect on the frequency of Lys⁺ revertants when UV treatment was followed by PR. Pol thus appears to prevent the accumulation of +1 frameshifts due to CPDs, but not to other types of UV-induced lesions. Although this is consistent with a role for Pol specifically in the error-free bypass of CPDs (JOHNSON et al. 1999), analysis of individual classes of frameshifts suggests that bypass of some CPDs by Pol can be mutagenic as well (see below). Finally, in either the presence or absence of PR, the induction of Lys⁺ revertants was greatly reduced in a rev3 background, indicating a role for Pol in the production of the majority of +1 frameshifts initiated by either CPDs or 6-4PPs. Although examination of mutagenesis in the absence vs. presence of PR clearly implicates CPDs as the lesion responsible for most in vivo frameshift mutagenesis, it should be noted that these results cannot be used to infer the relative mutagenic potential of a single CPD vs. 6-4PP.

View larger version (19K): In this window In a new window Download PPT slide   FIGURE 3.— Contributions of CPDs and 6-4PPs to +1 frameshift mutagenesis. UV-induced reversion of the lys2A746 allele in WT (•), rev3 (), rad30 (), or rev3 rad30 (x) strains in the absence (A) or presence (B) of photoreactivation (PR) is shown. The UV-induced reversion frequencies were calculated by subtracting the frequency in the starting culture (0 J/m2) from that obtained after each specified UV dose. Error bars correspond to the standard deviation.

 

UV-induced reversion spectra in a WT background:

The lys2A746 allele reverts by acquiring a compensatory +1 frameshift within an 150-bp reversion window defined by stop codons in the alternative reading frames. Our previous analyses of spontaneous lys2A746 reversion have demonstrated that mutation spectra provide a wealth of information that cannot be discerned by simply measuring mutation rates (MINESINGER and JINKS-ROBERTSON 2005; SABBIONEDA et al. 2005). The spectra of lys2A746 reversion events as a function of UV dose were determined by sequencing 100 revertants isolated following irradiation with 0, 10, 40, or 60 J/m² of UVC (Figure 4). In the absence of UV irradiation, 84% (87/104) of the reversion events were simple +1 frameshifts, and >90% of these (81/87) were in homopolymer runs >3 nt (Figure 4A). The two longest runs in the reversion window were hotspots for the simple +1 events; 76% of the +1 events were in the 6A run and 9% were in the 5T run. The strong 6A:5T bias most likely reflects the positive correlation between run length and the frequency of DNA polymerase slippage (TRAN et al. 1997; GREENE and JINKS-ROBERTSON 2001). In addition to the predominant class of simple insertions, complex insertions (cins) in which the selected frameshift was accompanied by one or more base substitutions accounted for 6% of the spontaneous events. Finally, 6% of the revertants contained a 95- or 131-bp deletion with endpoints in 10- or 7-bp direct repeats, respectively. The large deletions were most evident among the spontaneous revertants and presumably reflect polymerase slippage during normal, replicative DNA synthesis.

View larger version (16K): In this window In a new window Download PPT slide   FIGURE 4.— UV-induced lys2A746 reversion spectra in a WT (RAD30) background. The sequence of the reversion window for the lys2A746 allele is shown; the position of the nucleotide deleted to create the lys2A746 allele is indicated by a dash and the nucleotides changed to extend the reversion window are indicated by lowercase letters (HARFE and JINKS-ROBERTSON 1999). Pluses and minuses indicate 1-nt insertions and 2-bp deletions, respectively; complex insertions (cins) are indicated above the sequence. Complex events above the 4A run that are followed by an asterisk (cins*) indicate events where the frameshift event could have occurred at either the 5T run or the 4A run. The number of events created by the deletion of 95 or 131 bp with endpoints in 10- or 7-bp direct repeats, respectively, is indicated as "large DEL" above each spectrum. The total number of revertants sequenced (n) for each strain is indicated. The spontaneous WT spectrum (0 J/m2) was published previously (HARFE and JINKS-ROBERTSON 2000).

 
Reversion of the lys2A746 allele was stimulated 24-fold following UV irradiation at a dose of 10 J/m² (see Figure 2B), and this was accompanied by a very striking reversal in the relative numbers of simple insertions in the 6A vs. 5T run (Figure 4B). Whereas there was an 8:1 bias in favor of simple insertions within the 6A run among spontaneous revertants, there was a 5:1 bias in favor of 5T run insertions among the UV-induced revertants. Irradiation with 40 J/m² of UV stimulated the Lys⁺ frequency 560-fold, and the UV-associated bias for simple insertions in the 5T rather than the 6A run persisted (Figure 4C). Relative to the simple insertions, an enhanced accumulation of complex insertions also began to be evident at the 40 J/m² UV dose, with complex insertions increasing to 13% of the total reversion events. The shift toward complex insertions within the reversion spectrum was much more evident at the 60 J/m² UV dose, with these events accounting for 29% (31/109) of the total UV-induced revertants sequenced (Figure 4D). Whereas the spontaneous complex events were scattered throughout the reversion window, the UV-induced events clustered at hotspots that presumably reflect the abundances of the underlying UV lesions, the efficiency of NER, and/or the relative efficiencies of the lesion bypass/tolerance mechanisms. To confirm that the complex events induced by 60 J/m² UV were dependent on the presence of Pol, a spectrum was also obtained from a rev3 mutant. As expected, complex events were virtually eliminated in the rev3 mutant (Figure 4E), with only one event being observed out of the 99 revertants sequenced. Finally, spectra were obtained after allowing time for photoreactivation to specifically reverse CPDs. PR was carried out after the 10- and 60-J/m² UV exposures, and the corresponding spectra are presented in Figure 4, F and G.

UV-induced reversion spectra in a rad30 background:

The UV-induced reversion spectra generated in the absence of Pol (rad30 mutant) are presented in Figure 5. As with the WT strain, spectra were obtained following irradiation with 0, 10, 40, or 60 J/m² of UVC and when the 10- and 60-J/m² doses were followed by photoreactivation. In the absence of UV, the lys2A746 reversion spectrum of the rad30 mutant (Figure 5A) was similar to that of the WT strain (Figure 4A), with a strong bias for simple events in the 6A relative to the 5T run, and only a small percentage of the total frameshifts being complex. The UV-induced spectra generated in the rad30 background (Figure 5, B–D) were strikingly different, however, from those generated in the presence of Pol (Figure 4, B–D). First, there were roughly equivalent numbers of UV-induced simple events in the 6A and 5T runs in the rad30 mutant, while events in the 5T run were much more frequent in the WT background. Second, there was a dramatic increase in the proportion of complex events in the rad30 mutant, with complex events outnumbering the simple events at each UV dose examined. As in the WT strain, the complex events in the rad30 mutant were dependent on the presence of Pol, as these events were not evident when REV3 was deleted (Figure 5E).

View larger version (16K): In this window In a new window Download PPT slide   FIGURE 5.— UV-induced lys2A746 reversion spectra in a rad30 background. See Figure 4 legend for details.

 

Comparison of WT and rad30 spectra:

To facilitate the analysis of the frameshift reversion data, Figure 6 presents the frequencies of the three most prominent classes of +1 frameshifts: simple events in the 6A run, simple events in the 5T run, and complex events occurring throughout the reversion window. The frequency of a given class of event was derived by multiplying the measured Lys⁺ reversion frequency by the percentage of the relevant event in the corresponding spectrum. Only the frequencies following exposure of the WT and rad30 strains to the 10-J/m² dose of UV are shown, as no additional insight was gained when the higher UV doses were examined. With respect to spontaneous Lys⁺ revertants, the frequencies of +1 events in 6A and 5T runs, as well as the frequencies of complex events, were indistinguishable in the WT and rad30 strains. Upon exposure of the WT strain to UV, the proportion of events that were complex did not increase significantly, but, as noted previously there was a striking reversal in the distributions of +1 frameshifts between the 6A and 5T runs. This translates into an 180-fold induction by UV of simple insertions in the 5T run, but only a 4-fold induction of insertions in the 6A run. This dramatic difference could reflect the relative abundances of the primary lesions at or near these runs, the relative efficiency of lesion removal by NER, and/or the propensity of a given lesion to generate +1 frameshifts as opposed to other types of mutations. If one assumes that the UV-induced lesions that lead to the frameshifts are within the runs, it is interesting to note that the underlying dipyrimidine lesions in the 6A run would be on the transcribed (noncoding) strand, while those in the 5T run would be on the nontranscribed (coding) strand. Transcription-coupled NER would be expected to result in more efficient repair of lesions on the transcribed than on the nontranscribed strand (FRIEDBERG et al. 1995), and this could at least partially account for the strong bias for UV-induced insertions to occur in the 5T run.

View larger version (24K): In this window In a new window Download PPT slide   FIGURE 6.— Frequencies of specific classes of +1 frameshift events in WT and rad30 strains. Solid bars correspond to the overall lys2A746 reversion frequencies, hatched bars to the frequencies of simple insertions in the 6A run, shaded bars to the frequencies of simple insertions in the 5T run, and open bars to the frequencies of complex events. The reversion frequency (and standard deviation) for each type of event was calculated by multiplying the total frequency by the percentage of the specific event in the correponding spectrum. The UV data were generated at a dose of 10 J/m2.

 
Whereas the distributions of the spontaneous mutation types in the WT vs. rad30 strain were indistinguishable, those produced following UV irradiation were very different. First, the strong bias for the UV-induced events in the WT strain to occur in the 5T run rather than in the 6A run was not present in the rad30 mutant. If one compares the frequencies of these events in the WT and rad30 strains, the frequency of the 5T run events was 5-fold less in the rad30 than in the WT strain, while the frequency of the 6A run events was unaffected by loss of Pol. The +1 events that occurred in the 5T run thus were largely dependent on the presence of Pol, while those occurring in the 6A run were not. The second notable difference between the WT and rad30 spectra was the very striking increase in the proportion of Pol-dependent complex frameshifts upon loss of Pol, from 8% in the WT strain to 59% in the rad30 mutant. This translates into a 380-fold induction of Pol-dependent complex events in the absence of Pol, but only a 33-fold induction in its presence. Thus, in a rad30 mutant, UV lesions that are normally bypassed by Pol are instead bypassed by Pol to give the signature complex frameshifts. We suggest that these data reflect an underlying hierarchy in the utilization of Pol vs. Pol in lesion bypass, which could be related to the nature of the underlying lesion, the local sequence context surrounding a lesion, and/or the location of a lesion on the coding vs. noncoding strand of LYS2. An important goal of future studies will be to identify the factors that regulate the use of Pol vs. Pol in lesion bypass.

Further insight into the Pol-dependent bypass of UV-induced lesions was obtained by calculating the frequencies of the individual classes of frameshifts derived from CPDs vs. 6-4PPs (Table 1). The frequency of a given event attributable to 6-4PPs was calculated by multiplying the UV + PR Lys⁺ frequency by the proportion of the event in the corresponding UV + PR reversion spectrum. The frequency of the event due to CPDs was then calculated by subtracting the UV + PR frequency from the UV – PR frequency. In the WT strain, the largest class of UV-induced frameshifts was the simple +1 events in the 5T run, and >90% of these were attributable to CPDs. CPDs were also the major contributor to the 5T run events in the rad30 mutant, but, as noted above, events in the 5T run no longer dominated the spectrum. Whereas CPDs and 6-4PPs were equal contributors to the complex frameshifts and the 6A run +1 events in the WT background, CPDs contributed more to these frameshifts than did 6-4PPs in the rad30 strain. Together, these data are consistent with the role of Pol in the nonmutagenic bypass of CPDs; in its absence, CPDs are more likely to produce +1 frameshifts.

View this table: In this window In a new window   TABLE 1 Frequencies of specific mutation types following exposure to 10 J/m2 of UVC

 
A more complicated picture of lesion bypass by Pol emerges if one compares the contributions of a given lesion to each specific class of frameshift in the WT vs. rad30 background. As noted above, for example, most of the 6A run events were generated in response to CPDs in the rad30 mutant, while CPDs and 6-4PPs were equal contributors in the WT strain. A closer examination of the 6A run data reveals not only that Pol had the expected effect of reducing the CPD-generated +1 events (the frequency of these events increased twofold in the rad30 mutant), but also that Pol unexpectedly promoted the occurrence of +1 events derived from 6-4PPs (the frequency of these events decreased twofold in the rad30 mutant). In relation to a possible participation by Pol in the "error-prone" bypass of 6-4PPs to generate +1 frameshifts, it has been suggested that the RAD30-dependent reversion of the arg4-17 nonsense allele, which reverts primarily via C-to-T transitions, likely reflects the mutagenic bypass of a 6-4PP (ZHANG and SIEDE 2002). In addition, Pol has been shown to be required for the mutagenic bypass of a site-specific (6-4)TT engineered into a duplex plasmid (BRESSON and FUCHS 2002), although a contradictory result was obtained using an in vivo gap-filling assay (GIBBS et al. 2005). The data presented here extend the mutagenic potential of 6-4PPs to include the production of some types of UV-induced frameshift events by Pol. This particular role of Pol must be relatively minor, however, as this TLS polymerase did not contribute to overall survival in the UV + PR experiments.

In contrast to the equivalent contributions of 6-4PPs and CPDs to the 6A run and complex events in a WT background, >90% of the events in the 5T run were derived from CPDs. As noted above, loss of Pol was accompanied by the expected increase in the CPD-associated 6A run events, but the frequency of the 5T run events decreased upon loss of Pol. Thus, in the context of the 5T run, Pol promotes rather than antagonizes the production of simple +1 events derived from the bypass of CPDs. That Pol can have opposing roles in the CPD-associated frameshift mutagenesis that occurs in closely linked homopolymer runs (i.e., the 6A and 5T runs) illustrates the importance of examining mutation spectra in conjunction with measuring mutation frequencies.

Conclusions:

The contributions of the yeast TLS polymerases Pol and Pol to survival and frameshift mutagenesis following UVC irradiation were examined using WT, rev3, rad30, and rev3 rad30 strains. The specific removal of the CPDs by PR further allowed an estimation of the relative contributions of CPDs vs. other types of UV-induced lesions, which correspond primarily to 6-4PPs, to overall survival and mutagenesis. Consistent with an induction of RAD30 transcription by UV, the relative contributions of Pol and Pol to survival were dose dependent, with Pol being more important at low UV doses and Pol at high doses. As previously reported, synergism was evident at high doses in the rev3 rad30 double mutant, suggesting functional redundancy of Pol and Pol. At low UV doses, however, deletion of REV3 was epistatic to that of RAD30, suggesting that there may be joint action of Pol and Pol in bypass of some types of UV lesion bypass. Finally, the relative toxicities of CPDs and 6-4PPs were influenced by which TLS polymerase was functional.

In terms of UV-induced mutagenesis, +1 and –1 frameshifts appeared to be induced to similar levels by UVC and to compose a significant proportion of the total induced mutations. In agreement with presumed error-free and error-prone bypass of UV-induced lesions by Pol and Pol, respectively, reversion frequencies generally were elevated in the rad30 mutant and reduced in the rev3 mutant. A more detailed assessment of the contribution of Pol to +1 frameshift mutagenesis, however, was obtained by examining UV-induced frameshift spectra in WT and rad30 strains. While Pol generally prevented frameshift mutagenesis, as expected of an error-free TLS polymerase, it also appeared to promote the occurrence of some specific classes of events. Finally, an analysis of Pol-dependent complex frameshifts suggests a hierarchy in the utilization of Pol vs. Pol in the bypass of UV-induced lesions, a hierarchy that likely will extend to the bypass of other types of lesions. The complexities uncovered by the mutagenesis data reported here underscore the need to exercise caution when using a limited data set to deduce the bypass specificities of TLS polymerases in vivo.

We thank John McDonald for providing the pJM82 plasmid used in the construction of the rad30 strain, Brian Harfe for experimental contributions in the early phases of this study, and the members of the S.J.-R. laboratory for comments on the manuscript. Finally, we thank Nayun Kim, Gray Crouse, and Wolfram Siede for helping to clarify our thinking concerning the relative contributions of CPDs and 6-4PPs to survival. This work was supported by grant R01 GM064769 to S.J.-R. from the National Institutes of Health. A.L.A. was partially supported by Graduate Division of Biological and Biomedical Sciences at Emory University and National Institutes of Health training grant T32 GM-08367.

ARMSTRONG, J. D., and B. A. KUNZ, 1990 Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast. Proc. Natl. Acad. Sci. USA 87: 9005–9009.[Abstract/Free Full Text]

ARMSTRONG, J. D., and B. A. KUNZ, 1992 Photoreactivation implicates cyclobutane dimers as the major promutagenic UVB lesions in yeast. Mutat. Res. 268: 83–94.[Medline]

BARBOUR, L., and W. XIAO, 2003 Regulation of alternative replication bypass pathways at stalled replication forks and its effects on genome stability: a yeast model. Mutat. Res. 532: 137–155.[Medline]

BAYNTON, K., A. BRESSWON-ROY and R. P. P. FUCHS, 1999 Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae. Mol. Microbiol. 34: 124–133.[CrossRef][Medline]

BRESSON, A., and R. P. FUCHS, 2002 Lesion bypass in yeast cells: Pol participates in a multi-DNA polymerase process. EMBO J. 21: 3881–3887.[CrossRef][Medline]

CHEN, C., and R. D. KOLODNER, 1999 Gross chromosome rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Nat. Genet. 23: 81–85.[Medline]

FRIEDBERG, E. C., G. C. WALKER and W. SIEDE, 1995 DNA Repair and Mutagenesis. ASM Press, Washington, DC.

FRIEDL, A. A., B. LIEFSHITZ, R. STEINLAUF and M. KUPIEC, 2001 Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae. Mutat. Res. 486: 137–146.[Medline]

GIBBS, P. E., J. MCDONALD, R. WOODGATE and C. W. LAWRENCE, 2005 The relative roles in vivo of Saccharomyces cerevisiae Pol , Pol , Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer. Genetics 169: 575–582.[Abstract/Free Full Text]

GIETZ, R. D., and R. A. WOODS, 2002 Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350: 87–96.[CrossRef][Medline]

GREENE, C. N., and S. JINKS-ROBERTSON, 1997 Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins. Mol. Cell. Biol. 17: 2844–2850.[Abstract]

GREENE, C. N., and S. JINKS-ROBERTSON, 2001 Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities. Genetics 159: 65–75.[Abstract/Free Full Text]

HARACSKA, L., S. PRAKASH and L. PRAKASH, 2002 Yeast Rev1 protein is a G template-specific DNA polymerase. J. Biol. Chem. 277: 15546–15551.[Abstract/Free Full Text]

HARFE, B. D., and S. JINKS-ROBERTSON, 1999 Removal of frameshift intermediates by mismatch repair proteins in Saccharomyces cerevisiae. Mol. Cell. Biol. 19: 4766–4773.[Abstract/Free Full Text]

HARFE, B. D., and S. JINKS-ROBERTSON, 2000 DNA polymerase introduces multiple mutations when bypassing spontaneous DNA damage in Saccharomyces cerevisiae. Mol. Cell 6: 1491–1499.[CrossRef][Medline]

HOEIJMAKERS, J. H., 2001 Genome maintenance mechanisms for preventing cancer. Nature 411: 366–374.[CrossRef][Medline]

JANS, J., W. SCHUL, Y. G. SERT, Y. RIJKSEN, H. REBEL et al., 2005 Powerful skin cancer protection by a CPD-photolyase transgene. Curr. Biol. 15: 105–115.[CrossRef][Medline]

JOHNSON, R. E., S. PRAKASH and L. PRAKASH, 1999 Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Pol. Nature 283: 1001–1004.

JOHNSON, R. E., L. HARACSKA, S. PRAKASH and L. PRAKASH, 2001 Role of DNA polymerase in the bypass of a (6–4) TT photoproduct. Mol. Cell. Biol. 21: 3558–3563.[Abstract/Free Full Text]

JOHNSON, R. E., S. L. YU, S. PRAKASH and L. PRAKASH, 2003 Yeast DNA polymerase is essential for error-free replication past thymine glycol. Genes Dev. 17: 77–87.[Abstract/Free Full Text]

KANNOUCHE, P., and A. STARY, 2003 Xeroderma pigmentosum variant and error-prone DNA polymerases. Biochimie 85: 1123–1132.[Medline]

KOZMIN, S. G., Y. I. PAVLOV, T. A. KUNKEL and E. SAGE, 2003 Roles of Saccharomyces cerevisiae DNA polymerases Pol and Pol in response to irradiation by simulated sunlight. Nucleic Acids Res. 31: 4541–4552.[Abstract/Free Full Text]

KOZMIN, S., G. SLEZAK, A. REYNAUD-ANGELIN, C. ELIE, Y. DE RYCKE et al., 2005 UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 102: 13538–13543.[Abstract/Free Full Text]

KUNZ, B. A., M. K. PIERCE, J. R. A. MIS and C. N. GIROUX, 1987 DNA sequence analysis of the mutational specificity of UV light in the SUP4-o gene of yeast. Mutagenesis 2: 445–453.[Abstract/Free Full Text]

LAWRENCE, C. W., 2002 Cellular roles of DNA polymerase and Rev1 protein. DNA Repair 1: 425–435.[CrossRef][Medline]

LAWRENCE, C. W., T. O'BRIEN and J. BOND, 1984 UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast. Mol. Gen. Genet. 195: 487–490.[CrossRef][Medline]

MCDONALD, J. P., A. S. LEVINE and R. WOODGATE, 1997 The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism. Genetics 147: 1557–1568.[Abstract]

MILLER, J. H., 1985 Mutagenic specificity of ultraviolet light. J. Mol. Biol. 182: 45–65.[CrossRef][Medline]

MINESINGER, B. K., and S. JINKS-ROBERTSON, 2005 Roles of RAD6 epistasis group members in spontaneous Pol-dependent translesion synthesis in Saccharomyces cerevisiae. Genetics 169: 1939–1955.[Abstract/Free Full Text]

NELSON, J. R., C. W. LAWRENCE and D. C. HINKLE, 1996 Deoxycytidyl transferase activity of yeast REV1 protein. Nature 382: 729–731.[CrossRef][Medline]

PAULOVICH, A. G., C. D. ARMOUR and L. H. HARTWELL, 1998 The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage. Genetics 150: 75–93.[Abstract/Free Full Text]

PRAKASH, S., and L. PRAKASH, 2000 Nucleotide excision repair in yeast. Mutat. Res. 451: 13–24.[Medline]

PRAKASH, S., and L. PRAKASH, 2002 Translesion DNA synthesis in eukaryotes: a one- or two-polymerase affair. Genes Dev. 16: 1872–1883.[Free Full Text]

RATTRAY, A. J., and J. N. STRATHERN, 2003 Error-prone DNA polymerases: when making a mistake is the only way to get ahead. Annu. Rev. Genet. 37: 31–66.[CrossRef][Medline]

RAVANAT, J. L., T. DOUKI and J. CADET, 2001 Direct and indirect effects of UV radiation on DNA and its components. J. Photochem. Photobiol. B 63: 88–102.[CrossRef][Medline]

ROUSH, A. A., M. SUAREZ, E. C. FRIEDBERG, M. RADMAN and W. SIEDE, 1998 Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability. Mol. Gen. Genet. 257: 686–692.[CrossRef][Medline]

SABBIONEDA, S., B. K. MINESINGER, M. GIANNATTASIO, P. PLEVANI, M. MUZI-FALCONI et al., 2005 The 9–1-1 checkpoint clamp physically interacts with Pol and is partially required for spontaneous Pol-dependent mutagenesis in Saccharomyces cerevisiae. J. Biol. Chem. 280: 38657–38665.[Abstract/Free Full Text]

SANCAR, G. B., 2006 Damage recognition by DNA photolyases, pp. 95–110 in DNA Damage Recognition, edited by W. SIEDE, Y. W. KOW and P. W. DOETSCH. Taylor & Francis, New York.

SCHAAPER, R. M., R. L. DUNN and B. W. GLICKMAN, 1987 Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain. J. Mol. Biol. 198: 187–202.[CrossRef][Medline]

SHERMAN, F., 1991 Getting started with yeast. Methods Enzymol. 194: 3–20.[CrossRef][Medline]

STELTER, P., and H. D. ULRICH, 2003 Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425: 188–191.[CrossRef][Medline]

SWANSON, R. L., N. J. MOREY, P. W. DOETSCH and S. JINKS-ROBERTSON, 1999 Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae. Mol. Cell. Biol. 19: 2929–2935.[Abstract/Free Full Text]

TAYLOR, J.-S., 2006 Structure and properties of DNA photoproducts, pp. 67–94 in DNA Damage Recognition, edited by W. SIEDE, Y. W. KOW and P. W. DOETSCH. Taylor & Francis, New York.

THOMA, F., 1999 Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair. EMBO J. 18: 6585–6598.[CrossRef][Medline]

TRAN, H. T., J. D. KEEN, M. KRICKER, M. A. RESNICK and D. A. GORDENIN, 1997 Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants. Mol. Cell. Biol. 17: 2859–2865.[Abstract]

WASHINGTON, M. T., R. E. JOHNSON, S. PRAKASH and L. PRAKASH, 1999 Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase . J. Biol. Chem. 274: 36835–36838.[Abstract/Free Full Text]

YU, S.-L., R. E. JOHNSON, S. PRAKASH and L. PRAKASH, 2001 Requirement of DNA polymerase for error-free bypass of UV-induced CC and TC photoproducts. Mol. Cell. Biol. 21: 185–188.[Abstract/Free Full Text]

ZHANG, H., and W. SIEDE, 2002 UV-induced TC transition at a TT photoproduct site is dependent on Saccharomyces cerevisiae polymerase in vivo. Nucleic Acids Res. 30: 1262–1267.[Abstract/Free Full Text]

Communicating editor: L. S. SYMINGTON

This article has been cited by other articles:

				J. E. Stone, G. E. Kissling, S. A. Lujan, I. B. Rogozin, C. M. Stith, P. M. J. Burgers, and T. A. Kunkel Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase {zeta} Nucleic Acids Res., June 1, 2009; 37(11): 3774 - 3787. [Abstract] [Full Text] [PDF]

				L. S. Waters, B. K. Minesinger, M. E. Wiltrout, S. D'Souza, R. V. Woodruff, and G. C. Walker Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance Microbiol. Mol. Biol. Rev., March 1, 2009; 73(1): 134 - 154. [Abstract] [Full Text] [PDF]

				X. Zhong, P. Garg, C. M. Stith, S. A. N. McElhinny, G. E. Kissling, P. M. J. Burgers, and T. A. Kunkel The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins Nucleic Acids Res., October 18, 2006; 34(17): 4731 - 4742. [Abstract] [Full Text] [PDF]

International Access Link

Online ISSN: 1943-2361  Print ISSN: 0016-6731
Copyright 2009 by the Genetics Society of America
phone: 412-268-1812   fax: 412-268-1813   email: genetics-gsa{at}andrew.cmu.edu