Fragile X syndrome, the most common cause of inherited mental retardation, is instigated by dynamic expansion of a d(CGG) trinucleotide repeat in the 5'-untranslated region of the first exon of the FMR1 gene, resulting in its silencing. The expanded d(CGG)(n) tract readily folds into hairpin and tetraplex structures which may contribute to the blocking of FMR1 transcription. In this work, we report that the cationic porphyrin 5,10,15,20-tetra(N-methyl-4-pyridyl)porphin (TMPyP4) effectively destabilizes in vitro the G'2 bimolecular tetraplex structure of d(CGG)(n) while it stabilizes the G'2 tetraplex form of the telomeric sequence d(TTAGGG)(2). Similarly to TMPyP4, the hnRNP-related protein CBF-A also destabilizes G'2 tetrahelical d(CGG)(n) while binding and stabilizing tetraplex telomeric DNA. We report that relative to each agent individually, successive incubation of G'2 d(CGG)(n) with TMPyP4 followed by exposure to CBF-A results in a nearly additive extent of disruption of this tetraplex form of the repeat sequence. Our observations open up the prospect of unfolding secondary structures of the expanded FMR1 d(CGG)(n) tract of fragile X cells by their exposure to low molecular size drugs or to proteins such as TMPyP4 or CBF-A.

Small polydisperse circular DNA (spcDNA) is a heterogeneous population of extrachromosomal circular molecules present in a large variety of eukaryotic cells. Elevated amounts of total spcDNA are related to endogenous and induced genomic instability in rodent and human cells. We suggested spcDNA as a novel marker for genomic instability, and speculated that spcDNA might serve as a mutator. In this study, we examine the presence of telomeric sequences on spcDNA. We report for the first time the appearance of telomeric repeats in spcDNA molecules (tel-spcDNA) in rodent and human cells. Restriction enzyme analysis indicates that tel-spcDNA molecules harbor mostly, if not exclusively, telomeric repeats. In rodent cells, tel-spcDNA levels are higher in transformed than in normal cells and are enhanced by treatment with carcinogen. Tel-spcDNA is also detected in some human tumors and cell lines, but not in others. We suggest, that its levels in human cells may be primarily related to the amount of the chromosomal telomeric sequences. Tel-spcDNA may serve as a unique mutator, through specific mechanisms related to the telomeric repeats, which distinguish it from the total heterogeneous spcDNA population. It may affect telomere dynamics and genomic instability by clastogenic events, alterations of telomere size and sequestration of telomeric proteins.

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.

Fragile X syndrome is caused by expansion of a d(CGG) trinucleotide repeat sequence in the 5' untranslated region of the first exon of the FMR1 gene. Repeat expansion is thought to be instigated by formation of d(CGG)(n)secondary structures. Stable FMR1 d(CGG)(n)runs in normal individuals consist of 6-52 d(CGG) repeats that are interrupted every 9-11 triplets by a single d(AGG) trinucleotide. By contrast, individuals having fragile X syndrome premutation or full mutation present >54-200 or >200-2000 monotonous d(CGG) repeats, respectively. Here we show that the presence of interspersed d(AGG) triplets diminished in vitro formation of bimolecular tetrahelical structures of d(CGG)(18)oligomers. Tetraplex structures formed by d(CGG)(n)oligomers containing d(AGG) interspersions had lower thermal stability. In addition, tetraplex structures of d(CGG)(18)oligomers interspersed by d(AGG) triplets were unwound by human Werner syndrome DNA helicase at rates and to an extent that exceeded the unwinding of tetraplex form consisting of monotonous d(CGG)(18). Diminished formation and stability of tetraplex structures of d(AGG)-containing FMR1 d(CGG)(2-50)tracts might restrict their expansion in normal individuals.

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