Lesion bypass in yeast cells: Pol eta participates in a multi-DNA polymerase process

Abstract

Replication through (6-4)TT and G-AAF lesions was compared in Saccharomyces cerevisiae strains proficient and deficient for the RAD30-encoded DNA polymerase eta (Pol eta). In the RAD30 strain, the (6-4)TT lesion is replicated both inaccurately and accurately 60 and 40% of the time, respectively. Surprisingly, in a rad30 Delta strain, the level of mutagenic bypass is essentially suppressed, while error-free bypass remains unchanged. Therefore, Pol eta is responsible for mutagenic replication through the (6-4)TT photoproduct, while another polymerase mediates its error-free bypass. Deletion of the RAD30 gene also reduces the levels of both accurate and inaccurate bypass of AAF lesions within two different sequence contexts up to 8-fold. These data show that, in contrast to the accurate bypass by Pol eta of TT cyclobutane dimers, it is responsible for the mutagenic bypass of other lesions. In conclusion, this paper shows that, in yeast, translesion synthesis involves the combined action of several polymerases.

Fig. 1. Determination of TLS in vivo. Double-stranded plasmids carrying a single adduct (triangle), located within a short heteroduplex region (hatched rectangles), are constructed as described in Materials and methods, and used for strand segregation analysis as shown in (A). The sequence context of the heteroduplex region is shown in Figure 2 for the different lesion bypass assays implemented in the present paper. In (B), an example of a filter containing colonies obtained following transformation of plasmid pKB-3TG in wild-type yeast cells is shown. The left and right images are autoradiographs obtained upon hybridization with the target (3TG probe) and marker (3TG+3 probe) oligonucleotides, respectively. Control colonies transformed with plasmids pKB-4TG, pKB-3TG+3 and pKB-3TG are shown in boxes. Plasmid pKB-4TG is derived from plasmid pKB-3TG containing an additional T residue within sequence 5′-TTTG, thus mimicking the +1 mutation induced by AAF adducts in this sequence context (see Figure 2). As expected, the colonies containing control plasmid pKB-3TG+3 light up with probe 3TG+3 only. Conversely, colonies containing control plasmids pKB-3TG and pKB-4TG light up strongly and weakly with the 3TG probe only, respectively. Among the colonies analyzed, seven colonies (circles with solid outline) hybridize with both target and marker strand probes, suggesting that TLS occurred during replication. Three colonies (circles with broken outline) hybridize with target strand probe only, suggesting that TLS occurred during a gap-filling event. These latter TLS events most likely occur during gap filling of excision tracks generated during mismatch repair and have been shown to exhibit the same genetic requirements as TLS events that occur during replication (Baynton et al., 1998; data not shown). All other colonies responding to probe 3TG+3 only are scored as damage-avoidance events. A single colony marked with an X failed to grow (no signal with either probe).

Fig. 2. Specific lesion bypass assays. The location of the adduct within the target strand and the sequence of the marker strand are shown for all three lesion bypass assays that are implemented in the present work. The damaged nucleotides are shown in bold. The sequences of the error-free and mutagenic TLS events that are detected for each assay are shown.

Fig. 3. Bypass of a (6–4)TT photoproduct in S.cerevisiae wild-type and rad30 mutant strains. Error-free and mutagenic TLS events are determined as described in Materials and methods. Mutagenic TLS (T→C transitions) is strongly reduced either when Pol η is absent (rad30Δ) or catalytically defective (rad30AA). Error-free TLS is unaffected in the Pol η deletion strain, but is significantly reduced in the catalytically defective Pol η strain as if the mutant protein is able to compete with the DNA polymerase that mediates error-free bypass. Over 1000 colonies were analyzed by hybridization in each strain.

Fig. 4. Model for the bypass of (6–4)TT and G-AAF lesions in S.cerevisiae. (A) (6–4)TT. Data presented here suggest that, in vivo, Pol η inserts G ‘incorrectly’, while another polymerase inserts A ‘correctly’ opposite the 3′-T of the (6–4)TT lesion. In a wild-type background, both events occur at similar frequencies: 60% of misinsertion versus 40% of error-free insertion. Following this insertion step, both replication intermediates are likely to be extended by Pol ζ, yielding mutagenic and error-free bypass products, respectively (see text for discussion). (B) G-AAF. Within the 3TG sequence context, G-AAF adducts are mostly bypassed in an error-free manner. However, the adduct may also trigger a slippage event in the primer strand during replication of three Ts located 5′ to the adduct, thus yielding a +T frameshift mutation (semi-targeted frameshift event). As inactivation of Pol η suppresses 75% of all bypass events, we suggest that Pol η is involved mainly at the insertion step, and Pol ζ at the extension steps, yielding both error-free and frameshift bypass events. In the absence of Pol η, yet another DNA polymerase appears to perform limited insertion, as shown by the thin arrow (see text for further details).