Regulation of Rep Helicase Unwinding by an Auto-inhibitory Subdomain

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2019 Jan 29

PMID 30690484

Citations 17

Authors

Monika A Makurath

Kevin D Whitley

Binh Nguyen

Timothy M Lohman

Yann R Chemla

Affiliations

Soon will be listed here.

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.

Citing Articles

Structural Basis for Dimerization and Activation of UvrD-family Helicases.

Chadda A, Nguyen B, Lohman T, Galburt E bioRxiv. 2024; .

PMID: 39282289 PMC: 11398504. DOI: 10.1101/2024.09.05.611425.

Subunit Communication within Dimeric SF1 DNA Helicases.

Nguyen B, Hsieh J, Fischer C, Lohman T J Mol Biol. 2024; 436(11):168578.

PMID: 38648969 PMC: 11128345. DOI: 10.1016/j.jmb.2024.168578.

Directing Uphill Strand Displacement with an Engineered Superhelicase.

Hall-Thomsen H, Small S, Gavrilov M, Ha T, Schulman R, Moerman P ACS Synth Biol. 2023; 12(11):3424-3432.

PMID: 37844274 PMC: 10661026. DOI: 10.1021/acssynbio.3c00452.

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Bobrovnikov D, Makurath M, Wolfe C, Chemla Y, Ha T J Am Chem Soc. 2023; 145(39):21253-21262.

PMID: 37739407 PMC: 10557133. DOI: 10.1021/jacs.3c05254.

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Whinn K, Xu Z, Jergic S, Sharma N, Spenkelink L, Dixon N Nucleic Acids Res. 2023; 51(7):3307-3326.

PMID: 36938885 PMC: 10123100. DOI: 10.1093/nar/gkad186.

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