Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia.
Fanconi anemia (FA) is a cancer-prone hereditary disease resulting from mutations in one of the 13 genes defining the FANC/BRCA pathway. This pathway is involved in the cellular resistance to DNA-cross-linking agents. How the FANC/BRCA pathway is activated and why its deficiency leads to the accumulation of FA cells with a 4N DNA content are still poorly answered questions. We investigated the involvement of ATR pathway members in these processes. We show here that RAD9 and RAD17 are required for DNA interstrand cross-link (ICL) resistance and for the optimal activation of FANCD2. Moreover, we demonstrate that CHK1 and its interacting partner CLASPIN that act downstream in the ATR pathway are required for both FANCD2 monoubiquitination and assembling in subnuclear foci in response to DNA damage. Paradoxically, in the absence of any genotoxic stress, CHK1 or CLASPIN depletion results in an increased basal level of FANCD2 monoubiquitination and focalization. We also demonstrate that the ICL-induced accumulation of FA cells in late S/G2 phase is dependent on ATR and CHK1. In agreement with this, CHK1 phosphorylation is enhanced in FA cells, and chemical inhibition of the ATR/CHK1 axis in FA lymphoblasts decreases their sensitivity to mitomycin C. In conclusion, this work describes a complex crosstalk between CHK1 and the FANC/BRCA pathway: CHK1 activates this pathway through FANCD2 monoubiquitination, whereas FA deficiency leads to a CHK1-dependent G2 accumulation, raising the possibility that the FANC/BRCA pathway downregulates CHK1 activation.
Guervilly
Jean-Hugues
JH
Equipe voie FANC/BRCA et Cancer, CNRS FRE2939, CEA LRC43V, Univ Paris-Sud, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France.
Macé-Aimé
Gaëtane
G
Rosselli
Filippo
F
D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004789:Enzyme Activation; D005199:Fanconi Anemia / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection; D054875:Ubiquitination
D052236:Fanconi Anemia Complementation Group D2 Protein; D034741:RNA, Small Interfering; D008156:Luciferases; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1