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

Mutagenic translesion DNA polymerase V (UmuD(2)C) 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(2)C complex, similar to 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(2)C-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/ATPS 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 degrees C (similar to 1 to 1.5 h), slow at 30 degrees C (similar to 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/C17. 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. Author summary Escherichia coli upregulates more than 40 genes as part of the DNA damage-induced SOS regulon, many of which are involved in DNA repair and cell division. However, three DNA polymerases, pols V, II, and IV, are also induced to rescue replication forks blocked at persisting template lesions. Pol V (UmuD(2)C), encoded by the UV mutagenesis genes (umuDC), is primarily responsible for the increase in UV-induced chromosomal mutagenesis. However, pol V is catalytically inert. Interaction with a RecA nucleoprotein filament (RecA*) and ATP is required to convert pol V to an activated mutasome complex, pol V Mut = UmuD(2)C-RecA-ATP. Here, we show that pol V Mut deactivates dynamically during DNA synthesis, and statically in the absence of synthesis. Activated and deactivated states are governed by a conformational switch that repositions RecA relative to UmuC. Switching rates are more rapid at 37 than at 30 degrees C, and do not require ATP hydrolysis. ATP (ATPS) binding plays two required regulatory roles: 1) it allows binding of pol V Mut to primer-template DNA; 2) it triggers the RecA-UmuC conformational switch that activates pol V Mut.