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

Abstract

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, ~ 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/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 .

Betulinic Acid Prevents the Acquisition of Ciprofloxacin-Mediated Mutagenesis in Staphylococcus aureus

“…To provide more insights into the inhibitory action of BA towards SOS response induced by ciprofloxacin, we evaluated whether the co-treatment with these agents could affect the cell size of S. aureus . As earlier explained, during the SOS response, there is a blockage on the cell cycle, in an attempt to repair DNA damage, resulting in an increase of the cell size [41]. Therefore, cells incubated with ciprofloxacin showed an increased cell size, when compared to the non-treated cells (Figure 3).…”

The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

“…A circular DNA template (90 nt) with 20 nt primer attached was used to prevent β from sliding off a primer-template (p/t) DNA end. When present in an activated state, pol V Mut WT bound to ATP (pol V Mut WT-ATP) is barely able to synthesize DNA in the absence of β (Figure 3B) [25,32]. Although a primer extension band corresponding to the addition of 1 nt is clearly visible on the gel, Compared to the wild-type mutasome containing ATP, the SOS constitutive pol V Mut E38K/∆C17-ATP synthesized DNA to a far greater extent.…”

“…(2) spatial, where UmuC is sequestered on the cell membrane in an insoluble form until it binds to UmuD 2 and enters the cytosol [31]; (3) internal, where pol V Mut has an intrinsic DNA-dependent ATPase that regulates the binding of the mutasome to p/t DNA [25]; (4) conformational [32,33], where pol V Mut exists in activated and deactivated forms depending on the location of RecA in relation to UmuC and UmuD 2 . Two reviews describing the complex regulation of pol V Mut are contained in [34,35].…”

Antibiotic-Induced Mutagenesis: Under the Microscope

“…This process forms the mutagenically active complex pol V Mut (UmuD' 2 C-RecA-ATP; Jiang et al, 2009). Once formed, pol V Mut remains active for only a few seconds before cleaving the ATP (via intrinsic ATPase activity; an unprecedented function among polymerases; Erdem et al, 2014;Jaszczur et al, 2019), at which point the complex becomes inactive (Goodman et al, 2016).…”