Analyzing the Response to Dysfunction Replication Forks Using the RTS1 Barrier System in Fission Yeast.

ABSTRACT

The study of how eukaryotic cells overcome problems associated with dysfunctional DNA replication forks is assisted by experimental systems that allow site-specific replication fork arrest. Here we provide protocols for the use of the fission yeast RTS1 replication fork barrier. The RTS1 barrier is a directional, or polar, replication fork barrier that evolved to ensure directional replication of the fission yeast mating-type locus. We have moved the 859 bp RTS1 sequence to another locus in the genome and demonstrated that it arrests replication forks in a dysfunctional confirmation and that replication is restarted within ~20 min by the action of homologous recombination. We describe here the barrier constructs currently available, the methods by which we regulate the activity of the barrier, how to synchronize cells for analysis of replication intermediates by 2D gel electrophoresis, and the use of a replication slippage assay to measure fork fidelity.