FANCD2 Binds MCM Proteins and Controls Replisome Function upon Activation of S Phase Checkpoint Signaling

Abstract

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

PTEN regulates RPA1 and protects DNA replication forks

“…iPOND iPOND was performed as previously described [41,42]. PTEN +/+ and PTEN −/− HCT116 cells were labeled with 10 µM EdU for 15 min before 4 mM HU treatment for 5h.…”

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…113 An unbiased proteomics screen of chromatinbound FANCD2 immune complexes revealed that FANCD2 physically associates with the minichromosome maintenance 2-7 (MCM2-MCM7) replicative helicases. 114,115 FANCD2 interacts with MCM2-MCM7 in response to ATR-mediated DNA replication stress signaling independently of the core FA complex and monoubiquitination. 115 In the absence of FANCD2, replication forks stall stochastically and primary cells exhibit increased senescence, indicating that FANCD2 is required for replication fork progression.…”

“…114,115 FANCD2 interacts with MCM2-MCM7 in response to ATR-mediated DNA replication stress signaling independently of the core FA complex and monoubiquitination. 115 In the absence of FANCD2, replication forks stall stochastically and primary cells exhibit increased senescence, indicating that FANCD2 is required for replication fork progression. 115 Schlacher and colleagues also showed that FANCD2 is necessary to protect stalled replication forks from MRE11-mediated degradation, and that fork protection in the absence of FANCD2 can be rescued by RAD51.…”

“…115 In the absence of FANCD2, replication forks stall stochastically and primary cells exhibit increased senescence, indicating that FANCD2 is required for replication fork progression. 115 Schlacher and colleagues also showed that FANCD2 is necessary to protect stalled replication forks from MRE11-mediated degradation, and that fork protection in the absence of FANCD2 can be rescued by RAD51. 116 Moreover, it was recently discovered that FANCD2, in cooperation with BRCA1 and MRE11, recruits and interacts with CtIP (CtBPinteracting protein) at stalled replication forks to promote replication restart.…”

Replication stress and cancer: It takes two to tango

“…Besides its role in dealing with ICL, recent evidence has shown that the FA pathway might also be involved in safeguarding the integrity of replication forks. For instance, analysis of the composition of the replisome after treatment with hydroxyurea revealed the recruitment of FANCD2 through the interaction with phosphorylated MCM2, where it would slow down replication to allow fork stabilization [47]. FANCD2 has also been involved in the protection of reversed forks in cooperation with BRCA2 and Rad51 [34,48].…”