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. 1999 Aug 3;96(16):8919-24.
doi: 10.1073/pnas.96.16.8919.

V体育官网 - UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V

M Tang  1 , X Shen, E G Frank, M O'Donnell, R Woodgate, M F Goodman
Affiliations

UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V (V体育2025版)

M Tang et al. Proc Natl Acad Sci U S A. .

Abstract

The damage-inducible UmuD' and UmuC proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD'(2)C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD'(2)C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD'(2)C, the fifth DNA polymerase discovered in E. coli, be designated as E VSports手机版. coli pol V. In the presence of RecA, beta sliding clamp, gamma clamp loading complex, and E. coli single-stranded binding protein (SSB), pol V's polymerase activity is highly "error prone" at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the "A-rule," dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD'(2)C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3'-OH template-primer ends. We show, however, that despite competition for primer-3'-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion. .

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Figures

Figure 1
Figure 1
Separation of pol IIIts from the UmuD′2C complex by using Superdex 200 gel filtration. (Upper) Superdex 200 fractions (30 μl) containing pol III α subunit [designated as α (Left)] were resolved on an 8% SDS/PAGE gel and visualized by chemiluminescent immunodetection by using antiserum directed against α subunit. (Lower) Superdex 200 fractions (20 μl) containing UmuC protein [designated as C (Left)] were visualized on a 12% SDS/PAGE gel stained with Coomassie blue R-250. The presence of UmuC and UmuD proteins was verified by using UmuC and UmuD′ antisera (data not shown).
Figure 2
Figure 2
UmuD′2C (E. coli polV) has DNA polymerase activity. UmuD′2C was purified from a ΔpolB dnaE1026ts strain, RW588. Sephadex 200 fractions fx (50) containing predominantly pol IIIts; fx (56) containing pol IIIts + UmuD′2C and fx (64) containing UmuD′2C having no detectable pol IIIts were assayed for polymerase activity by extension of a 32P-labeled 30-mer primer annealed to a linear M13 DNA template, at permissive (37°C) and nonpermissive (47°C) temperatures. A single running-start base, C, is incorporated opposite G to reach the abasic lesion, X. The left-hand lane contains the primer (P) in the absence of proteins. Each reaction mixture contains 1.5 μl of the indicated fraction from a Superdex 200 gel filtration column (Fig. 1). All reactions were carried out in the presence of pol I antibody, RecA, SSB, β, γ complex, 4 dNTPs, and ATP.
Figure 3
Figure 3
Protein cofactor requirements for UmuD′2C (E. coli pol V)-catalyzed lesion bypass. Each reaction in lanes 1–9 contains 1.5 μl of Superdex 200 fx 64 containing UmuD′2C (having no detectable pol IIIts), pol I antibody, and various combinations of RecA, β, γ complex, and SSB, as indicated (lanes 1–9). Running-start reactions, in which C is incorporated opposite template G to reach the abasic site, were carried out at 37°C, with all four dNTPs present in lanes 1 to 8, but with dCTP omitted in lane 9. Reactions were run in the presence of 5% polyethylene glycol. A portion of the template sequence is shown at the right of lane 9, where X represents an abasic site. The left-hand lane contains the primer (P) in the absence of proteins. Standing-start reactions, in which the first incorporated nucleotide occurs opposite X, were run for wild-type UmuD′2C (lane 10) and for the mutant UmuD′2C104 (D101N) (lane 11), each at a concentration of 200 nM. A portion of the template, shown at the right of lane 11, has the same sequence as the running-start template, but uses a primer terminating one base before the lesion. The asterisk (*) designates a 32P-label at the 5′-primer terminus. The running-start primer-template DNA is shown at the top of Fig. 2.
Figure 4
Figure 4
Inhibition of UmuD′2C (E. coli pol V)-catalyzed translesion synthesis by wild-type pol III. A running-start C (20 μM dCTP) is incorporated opposite template G before reaching the abasic lesion, X. The concentration of dATP used for incorporation opposite X was varied to measure the kinetics of incorporation by either UmuD′2C, wild-type pol III, or a combination of both proteins. The UmuD′2C complex used in the reactions was purified from a strain containing wild-type pol III (18). Michaelis–Menten saturation plots of the translesion synthesis rate vs. dATP concentration are shown at the right. The translesion synthesis rate V is obtained by measuring IXΣ/IX−1 as a function of dATP concentration, where IXΣ are the integrated gel band intensities for incorporation at the site of the lesion and beyond, and IX−1 is the integrated gel band intensity at the G site, before reaching the lesion (see Materials and Methods). (A) Translesion synthesis catalyzed by UmuD′2C, pol III core HE, or combinations of both. (B) Translesion synthesis catalyzed by UmuD′2C, pol III α HE, or combinations of both. The dATP concentrations used were 0, 3, 10, 30, 100, 300, and 1,000 μM for A and 0, 2, 10, 50, 200, 500, and 1,000 μM for B. UmuD′2C is present at 200 nM in each experiment. The reactions were run at 37°C in the presence of RecA, SSB, and β, γ complex.
Figure 5
Figure 5
Kinetic analysis of the effect of wild-type pol III on UmuD′2C (E. coli pol V)-catalyzed translesion synthesis. Lineweaver–Burk double reciprocal plots for pol III HE and α HE (Upper) were generated from the kinetic data given in Fig. 4. Apparent Km and Vmax values and their ratios characterizing translesion synthesis by UmuD′2C (designated as Umu) in the absence and presence of pol III are provided in the tables below.
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