Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Abstract

Escherichia coli possesses three SOS-inducible DNA polymerases (Pol II, IV, and V) that were recently found to participate in translesion synthesis and mutagenesis. Involvement of these polymerases appears to depend on the nature of the lesion and its local sequence context, as illustrated by the bypass of a single N-2-acetylaminofluorene adduct within the NarI mutation hot spot. Indeed, error-free bypass requires Pol V (umuDC), whereas mutagenic (-2 frameshift) bypass depends on Pol II (polB). In this paper, we show that purified DNA Pol II is able in vitro to generate the -2 frameshift bypass product observed in vivo at the NarI sites. Although the Delta polB strain is completely defective in this mutation pathway, introduction of the polB gene on a low copy number plasmid restores the -2 frameshift pathway. In fact, modification of the relative copy number of polB versus umuDC genes results in a corresponding modification in the use of the frameshift versus error-free translesion pathways, suggesting a direct competition between Pol II and V for the bypass of the same lesion. Whether such a polymerase competition model for translesion synthesis will prove to be generally applicable remains to be confirmed.

Figure 1

Primer elongation experiments by Pol II on AAF- and UV-damaged DNA templates. The substrates used in this set of experiments consisted of a 24-mer primer (5′-³²P labeled) annealed to 70-mer templates. Templates contained either no lesion, single AAF in various sequence contexts (as shown), or a single T(6–4)T photoproduct. The 3′-end of the primer is located several nucleotides upstream from the lesion, as indicated. All reactions were performed under the standard reaction conditions (see Material and Methods) by using 1 nM primer/template coated with SSB (1 molecule of SSB per nucleotide) and 10 nM of Pol II (0.03 units). Reactions were performed at 30°C for 5 min, quenched with 20 mM EDTA, and analyzed by 12% denaturing polyacrylamide gels (8 M urea). Presence or absence of the lesion is symbolized by AAF or 0, respectively, whereas + and − indicate, respectively, the presence or absence of a given polymerase. Substrate structure is shown on top. (A) AAF adduct within the NarI (GGCG^AAFCC) sequence. (B and C) AAF adduct within sequence G₁G₂G₃, on G₁ or G₃. (D) Template with a T(6–4)T photoproduct.

Scheme 1

Figure 2

LT extension reactions. (A) The primer/template (20 mer/90 mer) used in this set of experiments mimics the LT at the NarI site, as the 3′-end of the primer is located across from the lesion site. (B) Reaction and analysis conditions as described in Fig. 1. Amount of polymerases, Klenow fragment exo− and Pol II exo+ and Pol II exo− were adjusted to provide the same efficiency of elongation on the nondamaged template for direct comparison 0.03 units of Pol II, 0.09 units of Pol II exo−, and 0.05 units of Klenow fragment exo−.

Figure 3

Replication intermediates formed during the extension of the NarI LT by Pol II. (A) Structure of the primer-template sequence is shown on top. Pol II (2 nM) and 1 nM SSB-coated primer/template (20 mer/70 mer) were incubated and analyzed under standard conditions in the presence of one or two dNTPs (100 μM each), as indicated. (B) Extension intermediates on unmodified and AAF-modified NarI templates. Structure of the potential replication intermediates taking into account the preferential incorporation at the +1 position of C and G with the AAF-modified and unmodified templates, respectively.

Figure 4

Extent of error-free and −2 frameshift TLS events in vivo. Error-free and −2 frameshift TLS events measured in vivo in different SOS-induced strains: wt, wild type strain MGZ; ΔpolB, strain MGZΔpolB; ΔpolB + pWKS130, strain MGZΔpolB transformed with pWKS130. ΔpolB + pWKS-polB⁺: strain MGZΔpolB transformed with the corresponding polB+ gene-expressing plasmid.

Figure 5

Model for TLS pathways at an AAF adduct located within the NarI site. Two distinct pathways for the bypass of a single AAF adduct located within the NarI site can be defined. The nonslipped pathway yielding error-free TLS requires the SOS-inducible DNA Pol V and RecA* similarly to UV-induced base substitution mutagenesis pathway. In contrast, the slipped pathway that generates −2 frameshift mutations requires another SOS-inducible DNA polymerase, namely DNA Pol II. Both nonslipped and slipped pathways are of similar importance in a wild-type strain representing 17 and 10% of the replication events, respectively. In vivo and in vitro studies allow us to describe the individual steps involved in both pathways. Formation of the LT by insertion of C opposite the G-AAF adduct appears to be feasible by either Pol III or Pol V. This replication intermediate, common to both pathways, can adopt two conformations referred to as nonslipped and slipped conformations. The nonslipped conformation exhibits a distorted 3′-end of the primer, whereas in its slipped conformation, the primer forms two correct GC base pairs at its extremity. In vivo analysis of TLS reveals a competition between Pol V and II for the elongation of the nonslipped and slipped lesion termini, respectively.