Replication fork collapse in vitro using Xenopus egg extracts

Abstract

When a replication fork encounters a nick in the parental DNA, the replisome dissociates and the replication fork structure is lost. This outcome is referred to as replication fork "collapse." Collapsed forks can be highly cytotoxic and mutagenic if not appropriately repaired by the cell. However, the events that occur during and after replication fork collapse are unclear. Here, we describe an in vitro system to induce site specific, strand specific replication fork collapse using Xenopus egg extracts, which contain the full set of DNA replication and repair enzymes. We also describe simple assays to monitor the stability of DNA nicks and the different structures formed during replication fork collapse. This methodology permits detailed mechanistic analysis of collapsed forks in vitro.

Keywords: Break-induced replication; DNA repair; DNA replication; Fork collapse; Fork stalling.