Error-free DNA repair initiated at the sites of replication fork stalling is critical to the prevention of genomic instability in cycling cells. Defects in stalled fork repair have been directly implicated in cancer and other human diseases. Fanconi Anemia (FA) is a rare, autosomal recessive (or X-linked) disease caused by inactivation of any one of several FA genes. The clinical manifestations of FA include childhood anemia and progressive bone marrow failure, together with short stature and congenital defects affecting a wide variety of organs. The risk of cancer, including solid tumors, is elevated, with particularly high incidence of acute myelogenous leukemia. The gene encoding a nuclease-coordinating scaffolding protein, SLX4/FANCP, is found mutated in some individuals with Fanconi anemia and has been implicated in stalled fork repair through interactions with the nucleases MUS81, XPF and SLX1. We adapted the Escherichia coli Tus/Ter replication fork arrest complex for use in mammalian cells and have used it to provoke site-specific replication fork stalling and homologous recombination (HR) at defined loci of a mammalian chromosome. We find that SLX4 plays a crucial role in mediating error-free HR induced by Tus/Ter. This function is restricted to stalled fork repair and is not a feature of HR induced by a conventional chromosomal double strand break. In work proposed here, we will use novel tools developed by the Scully lab, to analyze how SLX4 regulates homologous recombination at stalled replication forks. We will use physical and genomic assays to measure specific DNA structures that form at the Tus/Ter-stalled fork and will determine whether SLX4 regulates the formation or metabolism of these DNA structures. This project will identify the mechanisms by which SLX4 coordinates stalled fork processing to preserve genome stability in the face of replication stress. Success in this work will lead to the identification of new targets for therapy in cancer and other human diseases.
Every time a cell divides, it must duplicate the DNA that contains the blueprint of the cell. If the DNA replication process stalls at sites of damaged DNA, the cell must bring to bear an exquisitely tuned machinery of DNA repair and other activities to prevent DNA damage, mutation and cancer. In work described here, we will use powerful new tools, developed by the Scully lab, to study how damaged replicating DNA is repaired and to use those insights in an effort to discover new targets for cancer therapy.
