Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places

Electrophoresis
A J van BrabantB J Brewer

Abstract

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

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Citations

Dec 17, 2009·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·M K Raghuraman, B J Brewer
Jan 26, 2013·Genes & Development·Oscar M Aparicio
May 5, 2001·Molecular Cell·A J van BrabantB J Brewer
Jul 27, 2002·Annual Review of Microbiology·G T Marczynski, L Shapiro
Sep 25, 2003·The Journal of Biological Chemistry·Mark BowmakerIan J Holt
May 18, 1999·Molecular and Cellular Biology·A J van BrabantB J Brewer

Related Concepts

Genome Mapping
DNA, Fungal
Saccharomyces cerevisiae
2D Polyacrylamide Gel Electrophoresis
Chromosomes, Fungal
Replication Origin

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