Fork pausing complex engages topoisomerases at the replisome

Abstract

Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS- TIPIN) act as a "molecular brake"and promote fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that the Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost fromthe replisome. The C terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not forDNAreplication checkpoint (DRC) activation. Wepropose that forks pause at proteinaceous RFBs through a "sTOP"mechanism ("slowing down with topoisomerases I-II"), which we showalso contributes to protecting cells from topoisomerase-blocking agents.