The role of UHRF1 in Fanconi anemia pathway

Fanconi Anemia (FA) is a genetic disease caused by mutations in any one of the identified 16 genes. The corresponding proteins are known as FA proteins and compose the FA pathway, which is critical for the cellular response to DNA inter-strand cross-links (ICLs). Many efforts have been drawn to understand the individual role of the FA proteins, yet the mechanism of the initiation of the pathway remains elusive, and in particular, no sensor protein for ICLs has been identified. The aims of this study are to identify such a putative factor and to investigate the role of this factor in DNA ICL repair. We have analyzed the DNA binding activity of two up-stream players in the FA pathway, i.e. FANCD2 and FANCI, which do not seem to be able to detect the damage directly. We then designed a novel biochemical purification strategy using biotin-labeled ICL DNA to isolate ICL interacting proteins from HeLa nuclear extract. By using mass-spectrometry following the purification, we identified an E3 ligase named UHRF1. We cloned, expressed and purified UHRF1 from Sf9 cells. In vitro data demonstrate that UHRF1 specifically and directly interacts with cross-linked DNA. In vivo experiments indicate that UHRF1 participates in the FA pathway, potentially by recruiting FANCD2 to the sites of DNA damage, and the reduction of cellular levels of UHRF1 by RNA interference (RNAi) sensitizes cells to MMC. We also identified the SRA domain as the region of UHRF1 that is responsible for DNA ICL binding, and the interference of this region results in defects in the cells in ICL repair. With the unique feature of ICL DNA binding, UHRF1 is identified as a new player in the FA pathway. Future clinical studies will help to determine if UHRF1 is indeed an FA gene of which mutations can generate phenotypes of the disease. Also, a better understanding of the molecular mechanisms underlying UHRF1 in the FA pathway will enable us to develop better and more targeted modes of cancer therapies.