Probing replication origins with a fork

Abstract

In 1968, Huberman and Riggs used DNA-fibre autoradiography to dem- onstrate that the replication of eukaryotic chromosomes occurs bi- directionally from multiple discrete sites that appear to be irregularly spaced along the chromosome t. Since then, the specific sequence requirements of prokaryotic and viral replication origins have been well documented. By contrast, the de- tailed analysis of initiation sites in more complex systems has pro- gressed little in over 20 years. This has begun to change recently with the development of various tech- niques that allow the precise location and specific sequences of origins to be assessed. Recent advances have been achieved using two-dimensional (2-D) gel electrophoretic techniques that physically separate replication intermediates from nonreplicating DNA, followed by DNA hybrid- ization to probe for particular sequences of interest 2,3. The 2-D gel technique of Brewer and Fangman 2 is a logical extension of the work of Bell and Byers 4, which showed that branched and linear forms of DNA could be separated by 2-D neutral/ neutral (N/N) agarose gel electro- phoresis. In this system, replication intermediates have characteristic mi- gration patterns that allow initiation sites to be mapped. A second 2.D gel technique (neutral/alkaline, N/A) 3, developed by Nawotka and Huberman, utilizes alkaline denatur- ing conditions to separate nascent and parental strands, allowing origins and fork direction to be mapped. In addition, a number of biochemical