[Show abstract][Hide abstract] ABSTRACT: Fanconi Anemia (FA) and Bloom Syndrome share overlapping phenotypes including spontaneous chromosomal abnormalities and increased cancer predisposition. The FA protein pathway comprises an upstream core complex that mediates recruitment of two central players, FANCD2 and FANCI, to sites of stalled replication forks. Successful fork recovery depends on the Bloom's helicase BLM that participates in a larger protein complex ('BLMcx') containing topoisomerase III alpha, RMI1, RMI2 and replication protein A. We show that FANCD2 is an essential regulator of BLMcx functions: it maintains BLM protein stability and is crucial for complete BLMcx assembly; moreover, it recruits BLMcx to replicating chromatin during normal S-phase and mediates phosphorylation of BLMcx members in response to DNA damage. During replication stress, FANCD2 and BLM cooperate to promote restart of stalled replication forks while suppressing firing of new replication origins. In contrast, FANCI is dispensable for FANCD2-dependent BLMcx regulation, demonstrating functional separation of FANCD2 from FANCI.
Nucleic Acids Research 05/2013; 41(13). DOI:10.1093/nar/gkt348 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2-FANCI complex versus the monomeric proteins are. We show that the FANCD2-FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2-FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to-and independently of-FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase.
Nucleic Acids Research 06/2012; 40(17):8425-39. DOI:10.1093/nar/gks638 · 9.11 Impact Factor