Host cell RecA activates a mobile element-encoded mutagenic DNA polymerase.

Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA'2B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA'2B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.

Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP.

Mutagenic translesion DNA polymerase V (UmuD'2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD'2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD'2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is "rapid" at 37°C (~ 1 to 1.5 h), "slow" at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.

Insights into the complex levels of regulation imposed on Escherichia coli DNA polymerase V.

It is now close to 40 years since the isolation of non-mutable umu/uvm strains of Escherichia coli and the realization that damage induced mutagenesis in E.coli is not a passive process. Early models of mutagenesis envisioned the Umu proteins as accessory factors to the cell's replicase that not only reduced its normally high fidelity, but also allowed the enzyme to traverse otherwise replication-blocking lesions in the genome. However, these models underwent a radical revision approximately 15 years ago, with the discovery that the Umu proteins actually encode for a DNA polymerase, E.coli pol V. The polymerase lacks 3'→5' exonucleolytic proofreading activity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any "gratuitous" mutagenesis, the activity of pol V is strictly regulated in the cell at multiple levels. This review will summarize our current understanding of the myriad levels of regulation imposed on pol V including transcriptional control, posttranslational modification, targeted proteolysis, activation of the catalytic activity of pol V through protein-protein interactions and the very recently described intracellular spatial regulation of pol V. Remarkably, despite the multiple levels at which pol V is regulated, the enzyme is nevertheless able to contribute to the genetic diversity and evolutionary fitness of E.coli.

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.