Gel-Based Assays for Measuring DNA Unwinding, Annealing, and Strand Exchange

Methods Mol Biol. 2019:1999:255-264. doi: 10.1007/978-1-4939-9500-4_16.

Abstract

Efficient replication and repair of the genome requires a multitude of protein-DNA transactions. These interactions can result in a variety of consequences for DNA such as the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), the annealing of complementary ssDNAs, or the exchange of ssDNA with one strand of a dsDNA duplex. Some DNA helicases possess all three activities, but many DNA-interacting proteins can also catalyze one or more of these reactions. Assays that quantify these activities are an important first step in characterizing these protein-DNA interactions in vitro. Here, we describe methods for the formation of dsDNA substrates and the assays that can be used to biochemically characterize proteins that can unwind, anneal, and/or exchange DNA strands.

Keywords: DNA annealing; DNA helicase; DNA strand exchange; DNA unwinding; Radiolabeled DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Helicases / isolation & purification
  • DNA Helicases / metabolism*
  • DNA Replication*
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Enzyme Assays / methods*
  • Isotope Labeling / methods
  • Native Polyacrylamide Gel Electrophoresis / methods
  • Phosphorus Radioisotopes / chemistry
  • Protein Binding

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Phosphorus Radioisotopes
  • Phosphorus-32
  • DNA Helicases