Abstract
Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.
Supplemental material for this article may be found at http://mcb.asm.org/.
We thank Larry Thompson, Markus Grompe, Alan D’Andrea, Henri van de Vrugt, and Mathew Thayer for advice and many helpful discussions. We also thank James C. Bonner, Dietmar Gradl, and Doris Wedlich for providing us with XTC-2 cells. We are indebted to W. Dunphy for generously sharing antibodies for several Xenopus proteins and Jeff Parvin for sharing recombinant FA proteins.
A.S. received funding from the American Heart Association (0520117Z). V.C. and J.G. received funding from the National Institutes of Health (CA092245). K.A.C. received funding from the National Institutes of Health (GM62193). M.E.H. received funding from the Fanconi Anemia Research Fund, the Medical Research Foundation of Oregon, and the National Institutes of Health (CA112775).