Human DNA ligases I, III, and IV-purification and new specific assays for these enzymes

Methods Enzymol. 2006:409:39-52. doi: 10.1016/S0076-6879(05)09003-8.

Abstract

The joining of DNA strand breaks by DNA ligases is required to seal Okazaki fragments during DNA replication and to complete almost all DNA repair pathways. In human cells, there are multiple species of DNA ligase encoded by the LIG1, LIG3, and LIG4 genes. Here we describe protocols to overexpress and purify recombinant DNA ligase I, DNA ligase IIIbeta, and DNA ligase IV/XRCC4 and the assays used to purify and distinguish between these enzymes. In addition, we describe a fluorescence-based ligation assay that can be used for high throughput screening of chemical libraries.

Publication types

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

MeSH terms

  • Base Sequence
  • Catalytic Domain
  • DNA Ligase ATP
  • DNA Ligases / isolation & purification*
  • DNA Ligases / metabolism
  • DNA Probes
  • DNA Repair
  • Humans
  • Poly-ADP-Ribose Binding Proteins
  • Xenopus Proteins

Substances

  • DNA Probes
  • LIG1 protein, human
  • LIG4 protein, human
  • Poly-ADP-Ribose Binding Proteins
  • Xenopus Proteins
  • DNA Ligases
  • DNA Ligase ATP
  • DNA ligase III alpha protein, Xenopus
  • LIG3 protein, human