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Regulation of E. coli Rep helicase activity by PriC.
Nguyen, Binh; Shinn, Min Kyung; Weiland, Elizabeth; Lohman, Timothy M.
Affiliation
  • Nguyen B; Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States.
  • Shinn MK; Department of Physics, Washington University in St. Louis, St. Louis, MO 63130, United States.
  • Weiland E; Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States.
  • Lohman TM; Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States. Electronic address: lohman@wustl.edu.
J Mol Biol ; 433(15): 167072, 2021 07 23.
Article in En | MEDLINE | ID: mdl-34081984
Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: DNA Helicases / Escherichia coli Proteins / Escherichia coli Type of study: Prognostic_studies Language: En Journal: J Mol Biol Year: 2021 Type: Article Affiliation country: United States

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: DNA Helicases / Escherichia coli Proteins / Escherichia coli Type of study: Prognostic_studies Language: En Journal: J Mol Biol Year: 2021 Type: Article Affiliation country: United States