| dc.description.abstract | The linear ends of the eukaryotic chromosomes are protected by a specialized nucleoprotein complex known as telomere. Telomeres consist of telomere DNA and telomere DNA binding proteins (known as shelterin complex). The telomere DNA repeats consist of a 3’ G-rich overhang which folds into a lariat-like structure called the t/D-loops. The C-rich strand of telomere DNA is transcribed into a long non-coding RNA known as the telomere repeat containing RNA (TERRA) and comprises of repeats of UUAGGG in humans. Both TERRA RNA and telomere DNA, being G-rich, form higher order G-quadruplex structures both in vitro and in vivo. Thus, the higher order structures formed by telomere DNA along with TERRA RNA impart protection and stability to the telomere ends. However, these higher order structures have also been found to inhibit the telomere DNA elongation by the telomerase holoenzyme. Therefore, remodeling of the telomere DNA during its replication by DNA polymerase and elongation by the telomerase is important to prevent arrest of the replication. Several proteins are involved in t/D-loop disassembly and G-quadruplex unfolding in a non-enzymatic manner as well as using ATP dependent helicase activities.
In this thesis, we have studied two proteins, namely hnRNPA1 and RTEL1, involved in telomere DNA maintenance. hnRNPA1 is involved in unwinding of the G-quadruplex structures in a non-enzymatic fashion, whereas RTEL1 is involved in t/D-loop disassembly and G-quadruplex unfolding using its helicase activity. hnRNPA1 is a modular protein containing an N-terminal UP1 domain (comprising of two RNA Recognition Motifs, RRM1 and RRM2) followed by an RGG-box containing C-terminal region. The precise role of the RGG-box of hnRNPA1 in telomere DNA recognition and G-quadruplex DNA unfolding had remained unexplored. In this study, we showed that the RGG-box of hnRNPA1 is intrinsically disordered in solution and it specifically recognizes the TERRA RNA and telomere DNA G-quadruplex structures that have loops in their topology, whereas it shows no interaction with the single-stranded RNA/ DNA. Furthermore, the UP1 domain in the presence of the RGG-box destabilizes the loop containing TERRA RNA and telomere DNA G-quadruplexes efficiently compared to UP1 domain alone.
RTEL1 is a modular protein with four identified domains: N terminal Fe-S cluster containing DNA helicase domain, two harmonin-N-like domains (HNL1 and HNL2), a PCNA interacting protein motif box (PIP box), and a C terminal zinc binding C4C4 type RING finger domain. In this thesis, the sequential resonance assignment of the HNL1 and HNL2 domains has been achieved using triple resonance NMR methods and the initial structural characterization of these domains has been achieved with the help of CS-ROSETTA. . In addition, the interacting protein partners of Harmonin-N-like domains (tandem HNL1 and HNL2 domains) have been identified by pull down assays followed by mass-spectrometry analysis | en_US |
