Structural basis for unusual DNA conformations in CGG/CCG triplet repeats

• Main Authors: Yu-Wen Chen, 陳譽文
• Other Authors: 侯明宏
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/99588816402753167067

博士 === 國立中興大學 === 生物科技學研究所 === 102 === Some neurological diseases are correlated with expansion of (CCG)n / (CGG)n trinucleotide repeats, which contain many contiguous GpC flanked by mismatched C:C (G:G) base pair. Hairpin formation with the CGCC/CCGC stem leads to a C:C mismatched 5''-GC-3'' site, may enhance DNA slippage and is a possible factor for DNA expansion. According to the property of hairpin, it makes DNA to form We design a sequence In this various structures, including cruciform, triplex and tetraplex.We involving CCG repeats in order to study the d(TCCGCCGCCGA) structure.structure, tetraplex consists of two hairpin loop with i-motif in d(T(CCG)3A)2.We find that d(T(CCG)3A)2 is built by intercalation of two asymmetrical hairpin loops held together by four asymmetrical C*C pairs and by pair G4*G4, G10*G10 and A11*A11 with a CCG trinucleotide loop. The tailed thymine is stacked with asymmetric C6/G7 bases in the wide groove. Previous studies showed that a DNA groove-binding drug, chromomycin A3 (Chro), forms a dimer, mediated by a divalent metal ion, which binds to G/C-rich DNA, and CoII(Chro)2 is the better one to resist to spermine competition in the cells. First, we examine the effects of these MetalII(Chro)2 complexes on the CNG triplet repeats by analyzing the sequences containing various repeat number of CNG (N=A, T, C and G).It reveals that thermal stability of CoII(Chro)2 binding to CCG triplet repeats can increase higher with the repeat number than other triplet repeats. To characterize the binding of CoII(Chro)2 to CCG triplet repeats sequence, we solved the crystal structure of CoII(Chro)2-d(T(CCG)3A)2. The binding of CoII(Chro)2 to the minor groove of CCG triplet repeats cause nucleotide flipping out.Four G-specific hydrogen bonds between Chro and DNA provide the evidence of specific recognize of CoII(Chro)2. By using surface plasmon resonance assay, we obtained binding affinity of CoII(Chro)2 to CCG triplet repeats increased with repeat frequency. Moreover, we find that DNA offer a stable binding site when CCG repeat frequency of triplet repeat sequences achieves at least three times. On the other hand, we also want to explore the influence of CoII(Chro)2 binding to CGG repeat. The present study showed that GC sites with flanking G:G mismatches provide an excellent binding site for CoII(Chro)2 as shown by surface plasmon resonance and fluorescence analysis,compared to GC sites with flanking A:A, T:T, or C:C mismatches. We observed that CoII(Chro)2 could stabilize and trap the cruciform conformation of (CGG)16. Furthermore, two CoII(Chro)2 molecules may bind at the two GpC sites separated by at least one GpC site in the hairpin structure of (CGG)16.In a synthetic self-priming DNA model, 5’-(CGG)16(CCG)6-3’, CoII(Chro)2 can interfere with the expansion process of CGG triplet repeats, as shown by a gel electrophoretic expansion assay. Our findings present valuable structural information and experimental results for explanation and inhibition of the expansion of CCG/CGG triplet repeats. This study may provide potential applications of biological consequence of drug design in trinucleotide repeat diseases.