The processivity factor beta controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo

Abstract

The dinB-encoded DNA polymerase IV (Pol IV) belongs to the recently identified Y-family of DNA polymerases. Like other members of this family, Pol IV is involved in translesion synthesis and mutagenesis. Here, we show that the C-terminal five amino acids of Pol IV are essential in targeting it to the beta-clamp, the processivity factor of the replicative DNA polymerase (Pol III) of Escherichia coli. In vivo, the disruption of this interaction obliterates the function of Pol IV in both spontaneous and induced mutagenesis. These results point to the pivotal role of the processivity clamp during DNA polymerase trafficking in the vicinity of damaged-template DNA.

Fig. 1. Identification of the binding domain of Pol IV with the β-clamp by the two-hybrid system. (A) Description of the different Pol IV fragments used in the two-hybrid assays shown in (B). The five amino acids deleted in the constructed ΔC5 mutant appear in larger letters within the C16 fragment. (B) When the wild-type, C122, C16 or ΔC5 pGBKT7 constructs were co-transformed with the empty pGADT7 vector (-β in the figure), no growth occurred on the selective medium, indicating the absence of transcriptional auto-activation by these fusion proteins. When these constructs were co-transformed with the dnaN-containing pGADT7 vector (+β in the figure), growth on the selective medium was observed with wild-type, C122 and C16 pGBKT7 constructs, thus identifying the 16 last amino acids of Pol IV as containing the β-interaction domain of Pol IV. The lack of growth of the ΔC5-GBKT7/dnaN-pGADT7 co-transformants on selective medium suggests that the C-terminal pentapeptide of Pol IV is strictly required for efficient interaction.

Fig. 2. Effect of the β-clamp/Pol-IV interaction on its DNA polymerase activity in vitro. (A) DNA polymerase activity of Pol IV (200 fmol) and its ΔC5 derivative (200 fmol) were assayed on a 30/90 DNA substrate (20 fmol) in a 10 µl final volume for 1 min at room temperature as previously described (Wagner et al., 2000). Both enzymes exhibit a similar basal activity (lanes 2 and 6). An equivalent stimulation in activity is also seen upon addition of the SSB (1.22 pmol; lanes 3 and 7). However, while the presence of the β-clamp and the γ-complex (60 and 20 fmol, respectively) efficiently stimulates the activity of wild-type Pol IV (Wagner et al., 2000), no such increase is seen for the ΔC5 derivative (lanes 4 and 8). The structure of the primer template exhibits 3′ and 5′ single-stranded overhangs in order to prevent β from sliding off the template (Bloom et al., 1997). (B) DNA polymerase activity under the different experimental conditions is quantified and expressed as the amount of dNTP incorporated by 200 fmol of enzyme per minute. A 4-fold stimulation of the activity of both enzymes is found with SSB. In the presence of the β-clamp, the activity of wild-type Pol IV is increased an additional 4-fold, while the activity of the ΔC5 derivative remains unaffected.