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. 2000 Jan 18;97(2):565-70.
doi: 10.1073/pnas.97.2.565.

Highly Mutagenic Replication by DNA Polymerase V (UmuC) Provides a Mechanistic Basis for SOS Untargeted Mutagenesis

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Highly Mutagenic Replication by DNA Polymerase V (UmuC) Provides a Mechanistic Basis for SOS Untargeted Mutagenesis

A Maor-Shoshani et al. Proc Natl Acad Sci U S A. .
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Abstract

When challenged by DNA-damaging agents, Escherichia coli cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinsertion opposite the lesions, and an inducible mutator activity, which acts at undamaged sites. Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesion-bypass DNA polymerase, was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro reporter gene. This reaction required, in addition to pol V, UmuD', RecA, and single-stranded DNA (ssDNA)-binding protein. pol V produced point mutations at a frequency of 2.1 x 10(-4) per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymerase III holoenzyme. The mutational spectrum of pol V was dominated by transversions (53%), which were formed at a frequency of 1.3 x 10(-4) per nucleotide (1. 1% per cro gene), 74-fold higher than with pol III holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity). This finding suggests that replication by pol V, in the presence of UmuD', RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis.

Figures

Figure 1
Figure 1
Preparation of the gapped plasmid. Plasmid pOC2 (A) was nicked upstream to the cro gene with restriction enzyme AatII in the presence of ethidium bromide. This generated two subpopulations of nicked plasmid (B and C). Addition of exonuclease III extended the nicks into gaps in the 3′ → 5′ direction. Half of the molecules contain the cro gene in the single-stranded region.
Figure 2
Figure 2
Outline of the cro replication fidelity assay.
Figure 3
Figure 3
Gap-filling DNA replication by pol V and pol III holoenzyme. Gap-filling replication was performed with pol V (MBP-UmuC) in the presence of UmuD′, RecA, and SSB, or with pol III holoenzyme, by using gapped pOC2 as a substrate. Reactions were performed in parallel in the presence and the absence of radiolabeled dTTP at 37°C for 20 min. Reaction products were fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining (Left) or phosphorimaging (Right). Based on the analysis of the phosphorimage, the amount of radiolabel incorporated into DNA by pol V was 29.4% of the amount incorporated by pol III holoenzyme. FI, supercoiled plasmid; FII, open, circular plasmid; FIII, linearized plasmid; FIV, covalently closed and relaxed plasmid; GP, gapped plasmid.
Figure 4
Figure 4
Spectra of mutations generated in the cro gene during in vitro replication by pol V or by pol III holoenzyme. The 201 nt of the coding region of cro are shown. Mutations generated by pol V are shown above the cro sequence, whereas mutations generated by pol III holoenzyme are shown underneath the sequence. Δ, −1 deletion; M, −2 deletion; V, +1 insertion next to the marked nucleotide. The identity of the inserted nucleotide is shown in parentheses, unless it is identical to the template nucleotide after which it was inserted. W, +2 insertion. In addition to mutations in the coding region, eight mutations were found upstream to cro: for pol V, two C → A and one each of T → G, T → A, C → T, +T; for pol III holoenzyme, C → T and G → T, one each.

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