Robert M. Brosh, Jr.
National Institute on Aging, NIH, USA
Title: Analyses of patient-derived missense mutations in Fanconi anemia group J (FANCJ) DNA helicase
Biography
Biography: Robert M. Brosh, Jr.
Abstract
Statement of the Problem: Fanconi Anemia (FA) is a rare genetic DNA repair disorder characterized by progressive bone marrow failure, congenital abnormalities, and cancer. Of the 21 genes linked to FA, the FA Group J (FANCJ) gene is unique that it encodes an ATP-dependent DNA helicase. Mutations in FANCJ are not only genetically linked to FA, but also associated with breast and ovarian cancer. Consistent with its known role in homologous recombination (HR) repair, FANCJ-/- cells are sensitive to DNA interstrand cross-linking (ICL) agents and are also hypersensitive to agents that induce replication stress.
Methodology & Theoretical Orientation: We characterized two FA patient-derived FANCJ mutations, R707C and H396D, which reside in the conserved helicase core domain. Genetic and biochemical analyses were performed to delineate the molecular defects underlying the genetic disease.
Findings: FANCJ-R707C retained partial (~30%) helicase activity, whereas FANCJ-H396D was nearly completely inactive. Single-turnover kinetic assays, ATPase measurements, and DNA binding determinations confirmed the differential effects of FANCJ missense mutations on helicase activity. Expression of either FANCJ-R707C or FANCJ-H396D in fancj-/- cells completely failed to rescue cisplatin sensitivity. In striking contrast, expression of FANCJ-R707C in fancj-/- cells restored resistance to the DNA polymerase inhibitor aphidicolin, whereas FANCJ-H396D completely failed. Single-molecule replication tract analysis confirmed that FANCJ-R707C, but not FANCJ-H396D, restored fork rates after cellular exposure to aphidicolin. Thus, a quantitatively lower threshold of FANCJ catalytic activity is required for the aphidicolin-induced replication stress response compared to cisplatin-induced damage.
Conclusion & Significance: The catalytic requirement of FANCJ to reconstruct broken replication forks after ICL-induced damage is distinct from that required to remodel stalled replication forks. These findings provide new insight to FANCJ’s role in DNA repair and molecular phenotypes of clinically relevant FANCJ missense mutations that are relevant to human disease and cancer.