Purification and characterization of Escherichia coli DNA polymerase V

Methods Enzymol. 2006;408:378-90. doi: 10.1016/S0076-6879(06)08023-2.

Abstract

Cell survival and genome rescue after UV irradiation in Escherichia coli depends on DNA repair mechanisms induced in response to DNA damage as part of the SOS regulon. SOS occurs in two phases. The first phase is dominated by accurate repair processes such as excision and recombinational DNA repair, while the second phase is characterized by a large approximately 100-fold increase in mutations caused by an error-prone replication of damaged DNA templates. SOS mutagenesis occurs as a direct result of the action of the UmuDC gene-products, which form the low fidelity Escherichia coli DNA polymerase V, a heterotrimeric complex composed of UmuD'(2)C. This chapter describes the preparation of highly purified native pol V that is suitable for a wide range of biochemical studies of protein-protein, protein-DNA interactions and translesion-synthesis (TLS) mechanisms.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism
  • DNA Damage
  • DNA Repair
  • DNA-Directed DNA Polymerase* / genetics
  • DNA-Directed DNA Polymerase* / isolation & purification
  • DNA-Directed DNA Polymerase* / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli / radiation effects
  • Escherichia coli Proteins* / genetics
  • Escherichia coli Proteins* / isolation & purification
  • Escherichia coli Proteins* / metabolism

Substances

  • Escherichia coli Proteins
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Triphosphate
  • DNA polymerase V, E coli
  • DNA-Directed DNA Polymerase