title: Regulation of Mutagenic DNA Polymerase V Activation in Space and Time.
abstract: 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.
authors: Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors: Andrew
authors: A
authors: Robinson
authors: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors: John P
authors: JP
authors: McDonald
authors: Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors: Victor E A
authors: VE
authors: Caldas
authors: Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, California, United States of America.
authors: Meghna
authors: M
authors: Patel
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors: Elizabeth A
authors: EA
authors: Wood
authors: Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors: Christiaan M
authors: CM
authors: Punter
authors: Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors: Harshad
authors: H
authors: Ghodke
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors: Michael M
authors: MM
authors: Cox
authors: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors: Roger
authors: R
authors: Woodgate
authors: Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, California, United States of America.
authors: Myron F
authors: MF
authors: Goodman
authors: Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors: Antoine M
authors: AM
authors: van Oijen
mesh_terms: D004249:DNA Damage / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics; D013014:SOS Response, Genetics / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D014466:Ultraviolet Rays
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D011938:Rec A Recombinases; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase