Visualizing replication fork encounters with DNA interstrand crosslinks.

Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.