Molecular mechanisms for maintenance of G-rich short tandem repeats capable of adopting G4 DNA structures

Abstract

Mammalian genomes contain several types of repetitive sequences. Some of these sequences are implicated in various specific cellular events, including meiotic recombination, chromosomal breaks and transcriptional regulation, and also in several human disorders. In this review, we document the formation of DNA secondary structures by the G-rich repetitive sequences that have been found in several minisatellites, telomeres and in various triplet repeats, and report their effects on in vitro DNA synthesis. d(GGCAG) repeats in the mouse minisatellite Pc-1 were demonstrated to form an intra-molecular folded-back quadruplex structure (also called a G4′ structure) by NMR and CD spectrum analyses. d(TTAGGG) telomere repeats and d(CGG) triplet repeats were also shown to form G4′ and other unspecified higher order structures, respectively. In vitro DNA synthesis was substantially arrested within the repeats, and this could be responsible for the preferential mutability of the G-rich repetitive sequences. Electrophoretic mobility shift assays using NIH3T3 cell extracts revealed heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A3, which were tightly and specifically bound to d(GGCAG) and d(TTAGGG) repeats with K d values in the order of nM. HnRNP A1 unfolded the G4′ structure formed in the d(GGCAG) n and d(TTAGGG) n repeat regions, and also resolved the higher order structure formed by d(CGG) triplet repeats. Furthermore, DNA synthesis arrest at the secondary structures of d(GGCAG) repeats, telomeres and d(CGG) triplet repeats was efficiently repressed by the addition of hnRNP A1. High expression of hnRNPs may contribute to the maintenance of G-rich repetitive sequences, including telomere repeats, and may also participate in ensuring the stability of the genome in cells with enhanced proliferation. Transcriptional regulation of genes, such as c-myc and insulin, by G4 sequences found in the promoter regions could be an intriguing field of research and help further elucidate the biological functions of the hnRNP family of proteins in human diseases.