Quantitative and kinetic single-molecule analysis of DNA unwinding by Escherichia coli UvrD helicase.

Abstract

Helicases are nucleic acid-unwinding enzymes involved in the maintenance of genome integrity. Helicases share several “helicase motifs” that are highly conserved amino acid sequences and are classified into six superfamilies (SFs). The helicase SFs are further grouped into two classes based on their functional units. One class that includes SFs 3–6 functions as a hexamer that can form a ring around DNA. Another class that includes SFs 1 and 2 functions in a non-hexameric form. The high homology in the primary and tertiary structures among SF1 helicases suggests that SF1 helicases have a common underlying mechanism. However, two opposing models for the functional unit, monomer and dimer models, have been proposed to explain DNA unwinding by SF1 helicases. This paper briefly describes the classification of helicase SFs and discusses the structural homology and the two opposing non-hexameric helicase models of SF1 helicases by focusing on Escherichia coli SF1 helicase UvrD, which plays a significant role in both nucleotide-excision repair and methyl-directed mismatch repair. This paper reviews past and recent studies on UvrD, including the author's single-molecule direct visualization of wild-type UvrD and a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C), the latter of which was used in genetic and biochemical assays that supported the monomer model. The visualization revealed that multiple UvrDΔ40C molecules jointly unwind DNA, presumably in an oligomeric form, similar to wild-type UvrD. Therefore, single-molecule direct visualization of nucleic acid-binding proteins can provide quantitative and kinetic information to reveal their fundamental mechanisms.