Deficient regulation of DNA double-strand break repair in Fanconi anemia fibroblasts.
Fibroblasts from patients with Fanconi anemia (FA) display genomic instability, hypersensitivity to DNA cross-linking agents, and deficient DNA end joining. Fibroblasts from two FA patients of unidentified complementation group also had significantly increased cellular homologous recombination (HR) activity. Results described herein show that HR activity levels in patient-derived FA fibroblasts of groups A, C, and G were 10-fold greater than those seen in normal fibroblasts. In contrast, HR activity in group D2 fibroblasts was identical to that in normal cells. Western blot analysis revealed that the RAD51 protein was elevated 10-fold above normal levels in group A, C, and G fibroblasts, but was not altered in group D2 fibroblasts. HR activity levels in these former cells could be restored to near-normal levels by electroporation with anti-RAD51 antibody, whereas similar treatment of normal and complementation group D2 fibroblasts had no effect. These findings are consistent with a model in which FA proteins function to coordinate DNA double-strand break repair activity by regulating both recombinational and non-recombinational DNA repair. Interestingly, whereas positive regulation of DNA end joining requires the combined presence of all FA proteins thus far tested, suppression of HR, which is minimally dependent on the FANCA, FANCC, and FANCG proteins, does not require FANCD2.
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Sarah L
SL
Donahue
Richard
R
Lundberg
Rachel
R
Saplis
Colin
C
Campbell
D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D018274:Electroporation; D004926:Escherichia coli / Q000378:metabolism; D005199:Fanconi Anemia / Q000378:metabolism*; D052217:Fanconi Anemia Complementation Group A Protein; D052218:Fanconi Anemia Complementation Group C Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D052241:Fanconi Anemia Complementation Group G Protein; D051856:Fanconi Anemia Complementation Group Proteins; D005347:Fibroblasts / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009687:Nuclear Proteins / Q000502:physiology; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000502:physiology; D051135:Rad51 Recombinase; D011995:Recombination, Genetic
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C498471:FANCA protein, human; C497651:FANCC protein, human; C499905:FANCD2 protein, human; C499909:FANCG protein, human; C120277:Fanca protein, mouse; C497652:Fancc protein, mouse; C499904:Fancd2 protein, mouse; C499910:Fancg protein, mouse; D052217:Fanconi Anemia Complementation Group A Protein; D052218:Fanconi Anemia Complementation Group C Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D052241:Fanconi Anemia Complementation Group G Protein; D051856:Fanconi Anemia Complementation Group Proteins; D009687:Nuclear Proteins; D011506:Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse