High-resolution mapping of points of site-specific replication stalling

Methods Mol Biol. 2009:521:215-27. doi: 10.1007/978-1-60327-815-7_12.

Abstract

Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stalled replication fork gives a valuable insight into the causes and mechanisms of replication stalling. The method described here allows mapping of the position of the 3'-end of the nascent leading or lagging strand at the replication fork, stalled at a site-specific barrier. The replicating DNA is purified, digested with restriction enzymes, and enriched by BND-cellulose chromatography. The DNA is separated on a sequencing gel, transferred to a membrane, and hybridised to a strand-specific probe. The data obtained using this method allow determining the position of the 3'-end of the nascent strand at a stalled fork with a one-nucleotide resolution.

MeSH terms

  • Binding Sites / genetics
  • Chromatography, DEAE-Cellulose
  • DEAE-Cellulose / analogs & derivatives
  • DNA Replication / genetics*
  • DNA, Fungal / biosynthesis
  • DNA, Fungal / genetics
  • DNA, Fungal / isolation & purification
  • Electrophoresis, Polyacrylamide Gel
  • Molecular Probe Techniques
  • Nucleic Acid Hybridization
  • Nucleotide Mapping / methods*
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / growth & development
  • Schizosaccharomyces / metabolism

Substances

  • DNA, Fungal
  • benzoylated DEAE-cellulose
  • DEAE-Cellulose