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Regulation of mutagenic DNA polymerase V activation in space and time

Journal Article


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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


  •   Robinson, Andrew
  •   Mcdonald, John P. (external author)
  •   Caldas, Victor E. A. (external author)
  •   Patel, Meghna (external author)
  •   Wood, Elizabeth A. (external author)
  •   Punter, Christiaan M. (external author)
  •   Ghodke, Harshad
  •   Cox, Michael M. (external author)
  •   Woodgate, Roger (external author)
  •   Goodman, Myron F. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2015

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.

Scopus Eid


  • 2-s2.0-84940778030

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=4844&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/3821

Has Global Citation Frequency


Start Page


  • 1005482-1

End Page


  • 1005482-30

Volume


  • 11

Issue


  • 8

Place Of Publication


  • United States

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


  •   Robinson, Andrew
  •   Mcdonald, John P. (external author)
  •   Caldas, Victor E. A. (external author)
  •   Patel, Meghna (external author)
  •   Wood, Elizabeth A. (external author)
  •   Punter, Christiaan M. (external author)
  •   Ghodke, Harshad
  •   Cox, Michael M. (external author)
  •   Woodgate, Roger (external author)
  •   Goodman, Myron F. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2015

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.

Scopus Eid


  • 2-s2.0-84940778030

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=4844&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/3821

Has Global Citation Frequency


Start Page


  • 1005482-1

End Page


  • 1005482-30

Volume


  • 11

Issue


  • 8

Place Of Publication


  • United States