RESUMEN

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

Asunto(s)

Roturas del ADN de Doble Cadena , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Afidicolina/toxicidad , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Pollos , Cisplatino/toxicidad , ADN de Cadena Simple , Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , G-Cuádruplex , Mutación Missense , Oxazoles/toxicidad , ARN Helicasas/química , Recombinasa Rad51/análisis , Recombinasas/genética , Recombinasas/metabolismo , Proteína de Replicación A/metabolismo , Estrés Fisiológico