Eukaryotic DNA replication with purified budding yeast proteins

Methods Enzymol. 2021:661:1-33. doi: 10.1016/bs.mie.2021.08.018. Epub 2021 Oct 22.

Abstract

The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.

Keywords: Chromosome duplication; DNA replication; DNA replication assay; DNA replication in vitro; Protein expression; Protein purification; Reconstituted yeast DNA replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Replication
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism
  • Fungal Proteins / metabolism
  • Replication Origin
  • Reproducibility of Results
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Saccharomycetales* / genetics
  • Saccharomycetales* / metabolism

Substances

  • DNA, Fungal
  • Fungal Proteins
  • Saccharomyces cerevisiae Proteins