Elucidating the role of WRNIP1 in the protection of stalled replication forks

Abstract

DNA is the blueprint of life. It contains all the information necessary for the synthesis of proteins – the building blocks and workhorses of the cell. Maintenance of DNA in an unchanged state is therefore of utmost importance, since any mutations or loss of the information it holds may lead to catastrophic events, such as cell death or tumorigenesis. DNA needs to be faithfully copied with every cell division, which is an enormous task, given its size and importance. DNA replica- tion is constantly challenged by endogenous and exogenous factors, which have the potential to stall or stop replication – such a circumstance is called replication stress. While cells have evolved a number of mechanisms to deal with replication stress, e.g. checkpoint signaling that halts the cell cycle, DNA repair pathways that remove obstacles or factors that stabilise stalled replication forks, persisting replication stress inevitably leads to genomic instability and tumorigenesis. In- terestingly, cancer cells, due to their unrestricted proliferation, have elevated level of replication stress, which makes them more susceptible to anti-cancer therapies that exacerbate genomic in- stability. It is thus obvious that dissecting the events and mechanisms leading to and following replication stress is important from a basic science and clinical research point of view. Among the many aspects of replication stress, one that is gaining an increasing amount of at- tention is replication fork stability. Over the last years it was shown that a stalled replication fork is rapidly remodeled into a 4-way structure, an event that is believed to contribute to its stabil- isation. However, such a structure needs to be carefully maintained, since lack of its protection may cause an unscheduled processing by nucleases, leading to genomic instability. Interestingly, among the factors that protect stalled replication forks are many proteins previously associated with other DNA repair pathways, such as homologous recombination or Fanconi anaemia. The most recent member of the ’protectosome’ group is WRNIP1 (Werner helicase interacting protein 1), but its mode of action is unclear. In this project, we attempted to characterise WRNIP1’s biochemical activities and investigate further its cellular function. We have found that the protein exerts its protective function down- stream of replication fork reversal, challenging the current model. Our in vitro data show that WRNIP1 binds specifically to 4-way DNA junctions, a structure resembling a reversed replica- tion fork. We have also found that WRNIP1 interacts directly with the replication fork remod- eler ZRANB3 and that it is able to limit its replication fork reversal activity in vitro. Combined with published data, our data led us to propose a mechanism in which WRNIP1 binds to reversed replication forks immediately after their generation, thus protecting them against unscheduled MRE11-dependent degradation.