Abstract
Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases.
Highlights
Two Fanconi anemia patient missense mutations are localized in Fanconi anemia group J protein (FANCJ) helicase motif Ia
Western blot analysis demonstrated that R251C and Q255H were expressed at a similar level as the wild type (WT) (Fig. 1B)
We found that R251C and Q255H were recruited to the target area with the same kinetics as WT; the recruitment occurred within 5 min of photoactivation (Fig. 8C) and persisted without apparent diminution for at least 45 min
Summary
Two Fanconi anemia patient missense mutations are localized in FANCJ helicase motif Ia. R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA Both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. R251C abolished its ATP hydrolysis activity; Q255H contained normal DNA-dependent ATPase activity Both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or ␥-H2AX foci formation. Both mutant alleles exerted a domain negative effect when expressed in a wild-type background These results suggest that FANCJ helicase activity, but not ATPase or translocase activity, is required for FANCJ function in DNA repair
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