Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template.Author Summary: Escherichia coli, and many other bacteria, respond to high levels of DNA damage with an inducible system called the SOS response. In this response, bacteria first try to restart replication using non-mutagenic DNA repair strategies. If that fails, replication can be restored using DNA polymerases that simply replicate over DNA lesions, a desperation strategy that results in mutations. DNA polymerase V (pol V) is responsible for most mutagenesis that accompanies the SOS response. Because of the risk inherent to elevated mutation levels, pol V activation is tightly constrained. This report introduces a new layer of regulation on pol V activation, with a novel spatial component. After synthesis, the UmuC subunit of pol V is sequestered transiently at the membrane. Release into the cytosol and final activation depends on the activity of RecA protein and the autocatalytic cleavage of UmuD to generate the UmuD' subunit of pol V. The resulting delay in activation represents an additional molecular mechanism that limits the amount of time that this sometimes necessary but potentially detrimental enzyme spends on the DNA.