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PLoS Genet. 2019 Feb 4;15(2):e1007956. doi: 10.1371/journal.pgen.1007956. eCollection 2019 Feb.

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

Author information

1
Department of Biological Sciences, University of Southern California, Los Angeles, California, United States of America.
2
Department of Chemistry, University of Southern California, Los Angeles, California, United States of America.
3
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
4
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
5
Center of Applied Mathematical Sciences, University of Southern California, Los Angeles, California, United States of America.
6
Department of Physics and Astronomy, University of Southern California, Los Angeles, California, United States of America.

Abstract

Mutagenic translesion DNA polymerase V (UmuD'2C) 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'2C 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'2C-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.

PMID:
30716079
DOI:
10.1371/journal.pgen.1007956
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Conflict of interest statement

The authors have declared that no competing interests exist.

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