br Materials and methods br Results br Discussion G

Materials and methods
Results
Discussion G-quadruplex structures are extensively studied for its role in central dogma of molecular biology viz. replication problems leading to chromosomal translocations [20], [21], transcriptional inhibition [32] and alternative splicing and translational regulation [24], [25], [26], [27], [35] controlling protein synthesis. Since in each fusion gene in Xp11.2 translocation TFE3 is present however partners are changing therefore mechanism of translocation is of special interest at Xp11.2 locus and there may be some inherent property in TFE3 locus which make it fragile and exchanged with a variety of gene loci. Xp11.2 locus is repeat rich and involved in diseases like turner syndrome, neurodevelopmental disorders and autism by DNA rearrangement [37], [38], [39]. During replication and transcription DNA unwinds and before reannealing PQS can fold into a G-quadruplex structure (21), which further affect these processes. Replication blockage was studied by Taq NHS-SS-Biotin stoppage by PCR stop assay and if stretch of 4Gs was mutated to C this stoppage was inhibited showing G-quadruplex role in in vitro DNA amplification mimicking the DNA replication. Primer extension by Taq polymerase also showed the polymerase extension only upto G-quadruplex site. These studies show that how a stable G-quadruplex can be a threat for replication and if not resolved may lead to the DNA double strand break. Recent studies have shown that Stable G-quadruplexes are DNA replication fork barriers and has mutagenic consequences. If these structures are not resolved cause single strand DNA gaps leading to DNA double strand breaks in subsequent cell divisions [40], [41]. Non-B DNA structures of PKD1 are associated with genetic rearrangements (deletion and translocation) in close proximity of the mutations involved in human diseases [42], [43]. Bioinformatic study of lymphoid cancer shows a close association between occurrence of PQS and fragile region in 70% genes involved in rearrangements [18]. The whole Human genome is interspersed with sequences having potential to fold into various non-B DNA structures. Among these non-B DNA structures, the estimated average incidence of G-quadruplex forming sequence is 1 per 10,000 bases [44], [45]. Recent studies have shown that PQS with potential to fold into G-quadruplexes are present in promoters of the BCL2, MYC, KRAS, VEGF, KIT and HIF-α[21], regulating the gene expression. There are many DNA helicases which are melter of these stable G-quadruplex structures e.g. FANCJ, PIF1, REV1 [46], [47], [48] and those also regulate chromatin structure and epigenetic stability [49]. Thus logically these proteins could be used as therapeutics for the regulation of the aberrations caused by these stable G-quadruplexes [50]. Stability of these G-quadruplexes depends on temperature, G-quadruplex stabilizers and inhibitors. From reported study disruption of G-quadruplex structures by an antisense oligonucleotide complementary to the PQS was used to study the PQS behaviour. Melting temperature by melt curve analysis of dual labelled F23T probe was estimated at different temperatures in presence of PDS and ASO. PDS stabilizes PQS into G-quadruplex and increases the Tm and rescued towards the probe only Tm if added with ASO. Fusion point is mostly exon 5 and 6 and rare in exon 2, however G-quadruplexes are not the exact point of double strand break or chromosomal translocation. Stable G-quadruplexes formed in the break point region and nearby regions are also susceptible to genomic stability. Does G-quadruplex formation in the TFE3 explain its fragility during Xp11.2 translocation? Many reported studies have shown that non-B DNA structures (G-quadruplex, cruciforms and G-loops) are linked to chromosomal translocations [17], [18], [20], [21], [23]. Therefore a stable G-quadruplex in intron 2 alone or in association with other secondary structures in TFE3 might induce the DNA break and hence chromosomal translocation.