University of Wollongong
Browse

Regulation of mutagenic DNA polymerase V activation in space and time

Download (1.4 MB)
journal contribution
posted on 2024-11-15, 13:13 authored by Andrew Robinson, John P McDonald, Victor E A Caldas, Meghna Patel, Elizabeth A Wood, Christiaan M Punter, Harshad Ghodke, Michael M Cox, Roger Woodgate, Myron F Goodman, Antonius van OijenAntonius van Oijen
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.

History

Citation

Robinson, A., Mcdonald, J. P., Caldas, V. E. A., Patel, M., Wood, E. A., Punter, C. M., Ghodke, H., Cox, M. M., Woodgate, R., Goodman, M. F. & van Oijen, A. M. (2015). Regulation of mutagenic DNA polymerase V activation in space and time. PLoS Genetics, 11 (8), 1005482-1 - 1005482-30.

Journal title

PLoS Genetics

Volume

11

Issue

8

Language

English

RIS ID

107577

Usage metrics

    Categories

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC