Regulation of Rep helicase unwinding by an auto-inhibitory subdomain

Monika A. Makurath, Kevin D. Whitley, Binh Nguyen, Timothy M. Lohman, Yann Robert Chemla

Research output: Contribution to journalArticle

Abstract

Helicases are biomolecular motors that unwind nucleic acids, and their regulation is essential for proper maintenance of genomic integrity. Escherichia coli Rep helicase, whose primary role is to help restart stalled replication, serves as a model for Superfamily I helicases. The activity of Rep-like helicases is regulated by two factors: their oligomeric state, and the conformation of the flexible subdomain 2B. However, the mechanism of control is not well understood. To understand the factors that regulate the active state of Rep, here we investigate the behavior of a 2B-deficient variant (Rep2B) in relation to wild-type Rep (wtRep). Using a single-molecule optical tweezers assay, we explore the effects of oligomeric state, DNA geometry, and duplex stability on wtRep and Rep2B unwinding activity. We find that monomeric Rep2B unwinds more processively and at a higher speed than the activated, dimeric form of wtRep. The unwinding processivity of Rep2B and wtRep is primarily limited by ‘strand-switching’––during which the helicases alternate between strands of the duplex––which does not require the 2B subdomain, contrary to a previous proposal. We provide a quantitative model of the factors that enhance unwinding processivity. Our work sheds light on the mechanisms of regulation of unwinding by Rep-like helicases.

Original languageEnglish (US)
Pages (from-to)2523-2532
Number of pages10
JournalNucleic acids research
Volume47
Issue number5
DOIs
StatePublished - Jan 1 2019

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Optical Tweezers
Nucleic Acids
Maintenance
Escherichia coli
DNA

ASJC Scopus subject areas

  • Genetics

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Regulation of Rep helicase unwinding by an auto-inhibitory subdomain. / Makurath, Monika A.; Whitley, Kevin D.; Nguyen, Binh; Lohman, Timothy M.; Chemla, Yann Robert.

In: Nucleic acids research, Vol. 47, No. 5, 01.01.2019, p. 2523-2532.

Research output: Contribution to journalArticle

Makurath, MA, Whitley, KD, Nguyen, B, Lohman, TM & Chemla, YR 2019, 'Regulation of Rep helicase unwinding by an auto-inhibitory subdomain', Nucleic acids research, vol. 47, no. 5, pp. 2523-2532. https://doi.org/10.1093/nar/gkz023
Makurath, Monika A. ; Whitley, Kevin D. ; Nguyen, Binh ; Lohman, Timothy M. ; Chemla, Yann Robert. / Regulation of Rep helicase unwinding by an auto-inhibitory subdomain. In: Nucleic acids research. 2019 ; Vol. 47, No. 5. pp. 2523-2532.
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