Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork

Methods. 2016 Oct 1;108:65-78. doi: 10.1016/j.ymeth.2016.05.003. Epub 2016 May 9.

Abstract

Replicative helicases work closely with the replicative DNA polymerases to ensure that the genomic DNA is copied in a timely and error free manner. In the replisomes of prokaryotes, mitochondria, and eukaryotes, the helicase and DNA polymerase enzymes are functionally and physically coupled at the leading strand replication fork and rely on each other for optimal DNA strand separation and synthesis activities. In this review, we describe pre-steady state kinetic methods to quantify the base pair unwinding-synthesis rate constant, a fundamental parameter to understand how the helicase and polymerase help each other during leading strand replication. We describe a robust method to measure the chemical step size of the helicase-polymerase complex that determines how the two motors are energetically coupled while tracking along the DNA. The 2-aminopurine fluorescence-based method provide structural information on the leading strand helicase-polymerase complex, such as the distance between the two enzymes, their relative positions at the replication fork, and their roles in fork junction melting. The combined information garnered from these methods informs on the mutual dependencies between the helicase and DNA polymerase enzymes, their stepping mechanism, and their individual functions at the replication fork during leading strand replication.

Keywords: Bacteriophage T7 replication; Base pair unwinding-synthesis rate constant; Chemical step size; DNA polymerase; Mitochondrial DNA replication; Pre-steady state kinetics; Replicative helicase; Structural biology.

Publication types

  • Review
  • Research Support, N.I.H., Extramural

MeSH terms

  • 2-Aminopurine / chemistry
  • DNA Helicases / chemistry
  • DNA Helicases / genetics*
  • DNA Replication / genetics*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • Kinetics
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / genetics*
  • Protein Conformation

Substances

  • DNA-Binding Proteins
  • Multiprotein Complexes
  • 2-Aminopurine
  • DNA-Directed DNA Polymerase
  • DNA Helicases