Regulation of Rep helicase unwinding by an auto-inhibitory subdomain

• Published in: Nucleic acids research Vol. 47; no. 5; pp. 2523 - 2532
• Main Authors: Makurath, Monika A, Whitley, Kevin D, Nguyen, Binh, Lohman, Timothy M, Chemla, Yann R
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 18.03.2019
• Subjects: Adenosine Triphosphatases - genetics; DNA - chemistry; DNA - genetics; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Replication - genetics; DNA, Single-Stranded; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Kinetics; Models, Molecular; Mutation - genetics; Nucleic Acid Conformation; Nucleic Acid Enzymes; Protein Domains - genetics
• DOI: 10.1093/nar/gkz023
• ISSN: 0305-1048; 1362-4962

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 (RepΔ2B) 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 RepΔ2B unwinding activity. We find that monomeric RepΔ2B unwinds more processively and at a higher speed than the activated, dimeric form of wtRep. The unwinding processivity of RepΔ2B 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.