LB-231

Fanconi anemia (FA) is a genetic DNA repair disorder that results in chromosomal instability, bone marrow failure, birth defects, and cancer. To date, twelve genes have been identified that when mutated, lead to FA. Extensive research has shown that FA represents a novel DNA repair pathway that specializes in resolution of DNA interstrand crosslinks. The BRCA-1 Associated C-terminal Helicase (Bach1) is known to be the gene responsible for Fanconi anemia subtype J (FA-J), FANCJ. Previous data have shown that monoubiquitination of the FANCD2 protein in response to DNA damage and during S phase, considered to be an endpoint of the FA pathway, occurs in the absence of FANCJ. Consequently, there is speculation that FANCJ operates downstream of FANCD2 in the pathway. We are interested in defining the role of FANCJ within the FA pathway.

In this study we demonstrate that the FANCJ and FANCD2 proteins interact by immunoprecipitation, and confocal microscopy in HeLa cells and that only non-ubiquitinated FANCD2 binds to FANCJ. A mutant FANCD2 altered at the monoubiquitination site (K561R) co-immunoprecipitates FANCJ. Further, FANCJ and FANCD2 copurify in a 1.5MDa protein complex in undamaged cell extracts but, after DNA damage, separate into different protein complexes. Interestingly, FANCJ and FANCD2 interact only at G1/S of the cell cycle and not during S, G2, or M phase, as evidence by co-immunoprecipitation in synchronized HeLa cells. The FANCJ/FANCD2 interaction is dependent on an intact FA pathway and core complex, shown by immunoprecipitation of FANCJ and FANCD2 from FA-A mutant cell lines. However, FANCJ does not interact with the core complex, demonstrated by immunoprecipitation with FLAG-FANCA. Finally, FANCD2 chromatin localization and FANCD2 focus formation depend on FANCJ, as it is abrogated in FA-J mutant cells.
 >In summary, our results suggest that FANCJ does not function downstream of FANCD2 but rather interacts with and is responsible for loading FANCD2 onto chromatin following DNA damage. These studies serve to integrate FANCJ more tightly into FA biology and physically link the possibility of the helicase activity of FANCJ into the FA pathway.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA