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|Robert M Brosh, Sanjay Kumar Bharti, Irfan Khan, Joshua A and Sommers|
|National Institute on Aging, NIH, USA|
|ScientificTracks Abstracts: Transcriptomics|
|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 , 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- was nearly completely inactive. Singleturnover 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- 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- completely failed. Single-molecule replication tract analysis confirmed that FANCJ-R707C, but not FANCJ-, 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.|
Robert M Brosh has his expertise in DNA Repair and Genome Stability Maintenance. He leads a research group at the National Institute on Aging, NIH that is focused on characterizing the roles of clinically relevant human DNA helicases in cellular nucleic acid metabolism. This work has yielded insights into how DNA repair helicases promote phenotypes consistent with healthy aging and cancer resistance.
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