Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

• Published in: PLoS genetics Vol. 11; no. 8; p. e1005482
• Main Authors: Robinson, Andrew, McDonald, John P., Caldas, Victor E. A., Patel, Meghna, Wood, Elizabeth A., Punter, Christiaan M., Ghodke, Harshad, Cox, Michael M., Woodgate, Roger, Goodman, Myron F., van Oijen, Antoine M.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.08.2015; Public Library of Science (PLoS)
• Subjects: Bacteria; Chromosomes; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - genetics; DNA polymerase; DNA polymerases; DNA repair; DNA Replication - genetics; DNA, Bacterial - genetics; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; DNA-Directed DNA Polymerase - radiation effects; E coli; Enzyme Activation - genetics; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Escherichia coli Proteins - radiation effects; Funding; Gene Expression Regulation, Bacterial - genetics; Genetic aspects; Genetic regulation; Glucose; Light; Localization; Mutagenesis; Mutation; Observations; Physiological aspects; Protein Processing, Post-Translational - genetics; Rec A Recombinases - genetics; RecA protein; Regulation; SOS Response, Genetics - genetics; Transcription, Genetic - genetics; Ultraviolet Rays
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1005482

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.