Fanconi Anaemia (FA) is a rare inherited syndrome characterised by congenital abnormalities, progressive bone marrow failure and highly elevated risk of haematological and squamous cell cancers. The genetic pathway defective in FA is required for repair of DNA damage. Fifteen different “FANC” genes can cause FA, and seven of the gene products form the large ubiquitin ligase complex also known as the Fanconi core complex. Furthermore FANCD2 and FANCI are the substrates of this ubiquitin ligase.

The importance of ubiquitination to the repair of DNA damage is highlighted by the fact that at least 98% of FA patients show loss of the ubiquitinated form of FANCD2, and FANCD2-ubiquitination has been shown to be an essential step for the initiation of repair of specific DNA damage lesions. The biochemical mechanism of ubiquitination by the FA core complex is almost completely unknown, and why monoubiquitination is used as a signaling component also remains elusive.

We used the Multibac expression system to purify the entire intact FA core complex. Multibac allows simultaneous expression of the 9 proteins within the complex from a single baculovirus vector in insect cells. Affinity purification and size exclusion chromatography were used to isolate the complex for further analysis.

We have developed the first biochemical system to analyse the pathway defective in Fanconi anaemia in vitro, via reconstitution of the complexes involved in the ubiquitination and DNA repair steps.

We will present results relating to the purification, composition and ligase activity of the FA core complex expressed in the Multibac baculovirus system. A complete understanding of the biochemical mechanism of this multi-subunit ubiquitin ligase has implications for the understanding of DNA damage signaling in cancer therapy and FA patients.