Positive torsional strain causes the formation of a four-way junction at replication forks

The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)-D elta Lk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (+)-Delta Lk were not s upercoiled nor were the replicated arms interwound in precatenanes. The ele ctrophoretic mobility of these molecules indicated that they have no net wr ithe. Instead, the (+)-Delta Lk is absorbed by a regression of the replicat ion fork. As the parental DNA strands re-anneal, the resultant displaced da ughter strands base pair to each other to form a four-way junction at the r eplication fork, which is locally identical to a Holliday junction in recom bination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visual ized the structure with scanning force microscopy. We discuss possible phys iological implications of the junction for stalled replication in vivo.