The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E. coli pol III (ref. 4). Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis. (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis. Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand. The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites.

Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA synthesis including translesion synthesis (TLS). We have established three features of the roles of ATP and RecA. (1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase; (2) bound ATP is required for DNA synthesis; (3) pol V Mut function is regulated by ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase.DOI: http://dx.doi.org/10.7554/eLife.02384.001

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol switches to dNTP-stabilized misalignment. pol ␤ generates ؊1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.Pre-steady state kinetic studies of DNA polymerase fidelity have been focused on base substitution mutagenesis mechanisms (1, 2). Simple frameshift mechanisms have not yet been addressed despite the destructive biological consequences of having one or a few bases deleted or added. Small frameshifts, predominantly one-base deletions, are made on undamaged DNA by human DNA pol 1 , pol , pol ␤ (to a lesser extent) (3-7), and Escherichia coli pol IV (called simply "pol IV" throughout) (8 -10). Three models have been proposed to explain Ϫ1 frameshifts, namely the classical Streisinger model (11), direct misincorporation misalignment (3, 12), and dNTPstabilized misalignment (13, 14) (Fig. 1).Streisinger slippage results in simple deletions by displacement, i.e. the "looping out" of one or more bases as a primer strand slides along a run of reiterated template bases during replication (Fig. 1). Misincorporation misalignment occurs when DNA polymerase initially forms a mismatched base pair at the 3Ј-primer end that subsequently realigns by pairing with a complementary downstream template base prior to undergoing further extension (Fig. 1). Alternatively, DNA misalignment could occur as the first step followed by the "correct" incorporation of an incoming dNTP opposite a complementary downstream template base, a process referred to as dNTPstabilized misalignment (Fig. 1), which has been observed in the crystal structure of the pol IV homolog Sulfolobus solfataricus Dpo4 in ternary complex with DNA and an incoming nucleotide (15). The bottom line is that all three processes can follow different paths to arrive at the same mutational end point, a Ϫ1 deletion. Determining precise frameshifting mechanisms for individual DNA polymerases during replication and repair is an essential step toward understanding the basic principles of mutagenesis.In this study we perform pre-steady state fluore...

DNA polymerase fidelity is defined as the ratio of right (R) to wrong (W) nucleotide incorporations when dRTP and dWTP substrates compete at equal concentrations for primer extension at the same site in the polymerase-primer-template DNA complex. Typically, R incorporation is favored over W by 103 – 105, even in the absence of 3′-exonuclease proofreading. Straightforward in principal, a direct competition fidelity measurement is difficult to perform in practice because detection of a small amount of W is masked by a large amount of R. As an alternative, enzyme kinetics measurements to evaluate kcat/Km for R and W in separate reactions are widely used to measure polymerase fidelity indirectly, based on a steady-state derivation by Fersht. A systematic comparison between direct competition and kinetics has not been made until now. By separating R and W products using electrophoresis, we have successfully made accurate fidelity measurements for directly competing R and W dNTP substrates for 9 of the 12 natural base mispairs. We compare our direct competition results with steady state and presteady state kinetic measurements of fidelity at the same template site, using the proofreading-deficient mutant of Klenow Fragment (KF−) DNA polymerase. All the data are in quantitative agreement.

The ␤ protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the ␥ complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of ␤ onto DNA. Two ␤ dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5-O-(3-thiotriphosphate) (ATP␥S), have been used to show that ATP binding is required for ␥ complex and ␤ to associate with DNA, but that a ␥ complex-catalyzed ATP hydrolysis is required for ␥ complex to release the ␤⅐DNA complex and complete the reaction. In the presence of ATP and ␥ complex, the ␤ mutants associate with DNA as efficiently as wild type ␤. However, completion of the reaction is much slower with the ␤ mutants because of decreased ATP hydrolysis by the ␥ complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATP␥S in reactions using wild type ␤. Thus, the assembly of ␤ around DNA is coupled tightly to the ATPase activity of the ␥ complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.Replication of genomic DNA in Escherichia coli involves the assembly of a multisubunit enzyme complex on the DNA molecule. These proteins converge in such a way as to promote processive synthesis in both leading and lagging strands. In organisms as diverse as bacteriophages, bacteria, yeasts, and humans, the components that characterize a processive replicating machine are quite similar. In the case of E. coli, ␥ complex, using ATP, loads ␤ subunit onto DNA. The ␤ clamp, fully encircling the DNA molecule, associates with core and holds it firmly in place so extension of the primer can proceed. Because lagging strand synthesis is discontinuous, a cycling of the proteins upon completion of each Okazaki fragment to a new primer is required; therefore, periodic loading of ␤ is essential for replication of the genome to continue.Duplication of genomic DNA in E. coli requires processive DNA replication activity of the 10-subunit DNA polymerase III holoenzyme. The E. coli DNA polymerase III holoenzyme consists of three main functional units: the core polymerase, the ␤ sliding clamp, and the ␥ complex clamp loader (reviewed in Refs. 1 and 2). The three subunits distinctive to E. coli DNA polymerase III core (3) are ␣, which has DNA polymerase activity (4); ⑀, which has a 3Ј to 5Ј exonuclease activity (5, 6); and , which, at this time, lacks a well defined function (3, 7). The core, by itself, is not processive. It is capable of synthesizing short segments of DNA but dissociates readily from the primer/template, extending the primer only 10 -20 nucleotides per binding event in vitro (8). The ␤ sliding clamp and the ␥ complex are accessory proteins, the presence of which converts this nonp...