New mechanistic insight into replication fork reversal and restart

An emerging model of how stalled or damaged forks are processed is that replication fork s can reverse to aid repair of the damage. The first evidence that replication forks regress in human cells came from a recent study with topoisomerase I (T op 1) inhibitors, an important class of anticancer drugs currently in clinical use. Their cytotoxicit y, and thus their efficacy, has been generally linked to their ability to cause the accumulation of DNA nicks, which are later converted into double - stranded breaks (DSBs) by the collision of the DNA replication fork with the primary lesion. The discovery that replic ation forks can regress upon Top 1 inhibition provided new insight into the molecular basis of Top 1 cytotoxicity by showing that clinically relevant , nanomolar doses of Top 1 poisons induce replication fork slowing and reversal in a process that c an be uncoupled from DSB formation and requires poly(ADP - ribose) polymerase 1 ( PARP1) activity. However, w hether re versed forks can efficiently restart and wh ich factors are involved in this mechanism was still unknown. In this thesis , u sing a combination of biochemical and cellular approaches, we provided the first evidence that regressed forks can restart in vivo and identified a key role for the human RECQ1 helicase in promoting efficient re plication fork restart after Top 1 inhibition that is not shared by other human RecQ members . Our data also provided the first insight into the molecular role of PARP1 in fork reversal by showing that the poly(ADP - ribosyl)ation activity of PARP inhibits RECQ1 activity on replication forks after Top 1 inhibition. Thus, PARP activity is not required to form, but rather to "accumulate" reversed fork structures by maintaining/protecting them from a counteracting activity (RECQ1), which would otherwise cause an untimely restart of reversed forks, leading to DSB formation. The identification of a specific and controlled biochemical activity that drives the restart of reversed forks strongly supports the physiological relevance of this DNA transaction during replication stress in human cells. Moreover, our studies provide new mec hanistic insights into the roles of RECQ1 and PARP 1 in DNA replication and offer molecular perspectives to potentiate chemo therapeutic regimens based on Top 1 inhibition.