Distinct domains in the CArG-box binding factor A destabilize tetraplex forms of the fragile X expanded sequence d(CGG)_n

摘要

Formation of hairpin or tetraplex structures of the FMR1 gene d(CGG)_n sequence triggers its expansion, setting off fragile X syndrome. In searching for proteins that destabilize d(CGG)_n secondary structures we purified from rat liver quadruplex telomeric DNA binding protein 42 (qTBP42) that disrupts G'2 bimolecular tetraplex d(CGG)_n while paradoxically stabilizing the G'2 structure of the telomeric sequence d(TTAGGG)_n. Based on peptide sequence homology of qTBP42 and mouse CArG-box binding factor A (CBF-A), we provide direct evidence that recombinant CBF-A protein is physically and immunochemically indistinguishable from qTBP42 ant that it too destabilizes G'2 d(CGG)_n while stabilizing G'2 d(TTAGGG)_n. We inquired whether CBF-A employs the same or different domains to differentially interact with G'2 d(CGG)_n and G'2 d(TTAGGG)_n. Mutant CBF-A proteins that lack each or combinations of its five conserved motifs: RNP1_1, RNP1_2, RNP2_1, RNP2_2 and ATP/GTP-binding box were tested for their G'2 d(CGG)_n destabilization and G'2 d(TTAGGG)_n stabilization activities. We find that either RNP1_1 or the ATP/GTP motifs are necessary and sufficient for G'2 d(CGG)_n destabilization whereas RNP2_1 suppresses destabilization by either one of these two motifs. Neither RNP1_1 nor the ATP/GTP motif are required for G'2 d(TTAGGG)_n stabilization. Hence, CBF-A employs different domains to destabilize G'2 d(CGG)_n or stabilize G'2 d(TTAGGG)_n.