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

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Abstract
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 and 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 .