High-resolution mapping of points of site-specific replication stalling.
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.
Vengrova
Sonya
S
Marie Curie Research Institute, The Chart, Oxted, Surrey, UK.
Dalgaard
Jacob Z
JZ
D001665:Binding Sites / Q000235:genetics; D002848:Chromatography, DEAE-Cellulose; D003636:DEAE-Cellulose / Q000031:analogs & derivatives; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D009710:Nucleotide Mapping / Q000379:methods*; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
D004271:DNA, Fungal; C055068:benzoylated DEAE-cellulose; D003636:DEAE-Cellulose