| dc.description.abstract | Maintaining genomic integrity is indispensable for the survival and propagation of an organism. Failure to do so can cause mutations involving structural and functional aberrations, leading to severe diseases. The causative agent of TB, Mycobacterium tuberculosis (Mtb), is considered one of the most successful human pathogens. It is an intracellular pathogen that infects and multiplies within the host macrophages. Inside macrophages, the bacterium is exposed to various DNA-damaging agents. Additionally, endogenous factors such as by-products of many normal physiological processes and the extracellular environment, most commonly ultraviolet radiations, can also damage the DNA. The errors in replication and transcription, resulting in the incorporation of inappropriate bases, can also disrupt genomic integrity. The precursor molecules for DNA synthesis, i.e., the nucleotide pool, can also get altered and incorporated into the DNA. Furthermore, the G+C-rich genome (~ 64%) of Mtb makes it more susceptible to guanine oxidation and cytosine deamination. The pathogen's success lies in its ability to survive all these harsh conditions. Therefore, Mtb has developed various DNA-repair and error-avoidance mechanisms. The enzymes in these pathways recognize and rectify DNA damage and maintain genomic integrity. It is crucial to understand these, which may help design novel therapeutic approaches for controlling this disease.
The research described in this thesis involves the structural and functional characterization of uracil-DNA glycosylase (Ung) from Mtb and MutT1 from Mycobacterium smegmatis. A brief overview of the relevant literature on DNA damage and repair with emphasis on structural and biochemical studies of these two proteins is discussed in the introductory chapter. The first half of this chapter presents a brief account of DNA repair involving base excision, emphasizing uracil removal by uracil-DNA glycosylase. Details on the generation of oxidatively damaged nucleotides, their consequences, and sanitization by MutT proteins are provided in the latter half. Oxidative stress can cause modification of nucleotide bases in both the nucleotide pool and the DNA/RNA. Guanine is most susceptible to oxidation among all the nucleotides because of its low redox potential. Guanine oxidation at the 8th position forms 8-oxo-7,8-dihydroguanine (8-oxo-G), the most frequent among the oxidatively modified bases. 8-oxo-dGTP can be ambiguously incorporated against cytosine (C) and adenine (A) during DNA replication, resulting in G:C to T:A and A:T to C:G transversions, respectively. Additionally, direct oxidation of guanine in DNA forms an 8-oxo-G:C pair that, if not efficiently eliminated or repaired, can induce a G:C to T:A transversion. MutT proteins are a part of a three-component GO repair system that prevents mutations caused by 8-oxo-G. They belong to the Nudix hydrolase superfamily and catalyze the hydrolysis of substrates with a typical structure involving a nucleoside diphosphate linked to a moiety X (NDP-X) to NMP and P-X. Several previous studies have established an antimutator role of MutT proteins, i.e., strains lacking MutT leads to an increase in the frequency of mutations compared to the wild type. Mycobacterial MutT proteins, MutT1 and MutT2, have been shown to have an 8-oxo-Guanosine triphosphatase activity. Mycobacterium smegmatis MutT1 (MsMutT1) is a multifunctional two-domain enzyme. It consists of an N-terminal Nudix hydrolase domain and a C-terminal histidine phosphatase domain. The action of MsMutT1 towards Nudix substrates such as 8-oxo-dGTP, 8-oxo-GTP, and diadenosine polyphosphates has already been established. An exciting aspect of the mode of action of MsMutT1 is its modulation by the nature of the molecular interactions. This enzyme, which hydrolyses 8-oxo derivatives of guanosine triphosphate, does not act on GTP and dGTP under normal conditions. To further explore this aspect and elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of the enzyme complexes with 8-oxo-dGTP, GMPPNP, and GMPPCP were determined. This permitted a detailed comparison of the enzyme interactions with 8-oxo derivatives of guanosine triphosphates and with non-hydrolyzable analogs of GTP. This comparison led to further elucidation of the structural basis for the difference between the action of MsMutT1 on GTP and its 8-oxo derivatives. The work also gave insights into the correlation among intermolecular interactions, plasticity, and the activity of MsMutT1.
Uracil is a nucleotide base that is usually a component of RNA. However, it can erroneously get incorporated into DNA, which, if not corrected, can lead to mutations and interfere with the DNA binding proteins. Uracil can arise in DNA either by deamination of cytosine within DNA or by incorporating dUTP in DNA during replication. Uracil-DNA glycosylase is an important class of DNA repair enzymes that recognizes and catalyzes uracil excision from single-stranded and double-stranded DNA substrates and initiates the base-excision repair (BER) pathway. Molecular genetics studies have shown the importance of Ung in mycobacteria. The mutation rate substantially increases in the absence of this enzyme. Another study showed the importance of Ung in the survival of Mtb inside its host. A proteinaceous inhibitor (Ugi) encoded by Bacillus subtilis bacteriophage PBS1 or PBS2 is well known to inhibit UNG/Ung proteins. The bacteriophage expresses it as a part of the defense mechanism against host Ung. Structural studies showed that Ugi binds at the DNA-binding surface of UNG by mimicking the DNA backbone interactions, preventing UNG-DNA binding. MtUng has been well-characterized biochemically and structurally. The crystal structures of the native enzyme in various forms and in complex with different small molecules, namely, citrate, uracil, and uracil derivatives, are known. These structures comprehensively describe the uracil binding site and the extended binding site. The extended binding pocket involves the region which interacts with the sugars and phosphates of DNA. The element which leads to the enzyme's specificity is primarily its uracil binding pocket, making it an ideal target for drug design. The products formed from the Ung action on uracil-containing DNA are known to act as its inhibitors. The product, uracil, binds to the enzyme's active site and serves as its inhibitor. The enzyme is also inhibited to various extents by uracil analogs. Therefore, uracil-directed ligand tethering is an efficient strategy for inhibitor development of uracil-DNA glycosylase. We developed a molecular beacon-based fluorescence method to analyze the real-time action of MtUng on DNA substrates and its inhibition by small molecules. The method uses a hairpin oligo (5'-5-FAM-CUUUUUGAGCTTTTGCTCAAAAAG-BHQ-1-3') wherein five consecutive uracil residues in the stem region of the hairpin were incorporated. The oligomer was terminally attached with a commonly used fluorophore, 5-carboxyfluorescein (5-FAM), which is quenched by BHQ-1. Upon treatment with Ung, excision of the consecutive uracils results in the unwinding of the stem region of the oligomer to rapidly separate the quencher from the fluorophore yielding an intense fluorescence signal. We demonstrate the sensitivity of this method and its application in determining the efficiency of inhibition of MtUng by uracil derivative. The inhibition analysis by this method endorses high-throughput screening of compounds which can accelerate the process of drug discovery against infectious diseases by targeting their DNA-associated proteins.
To identify novel MtUng inhibitors, we systematically compiled and screened small molecule libraries in silico. The molecular docking and Molecular Mechanics Generalised Born Surface Area (MMGBSA) methodology were adopted for Structure-based virtual screening (SBVS), resulting in the identification of several hits. For experimental validation, 20 molecules were sourced and screened in vitro against MtUng, resulting in six hits with the half maximal inhibitory concentration (IC50) lower or equivalent to uracil. The results suggest that the probability of obtaining high MtUng inhibition increases with the presence of a uracil ring. Several uracil derivatives and compounds similar to uracil were also tested for inhibition. In this study, nineteen crystal structures accounting for twelve unique inhibitor complexes were determined. A network of conserved water molecules at the ligand-binding site was identified. This observation can be further used in the water mimic inhibitor design of MtUng.
MsMutT1 is a versatile multifunctional enzyme. Based on structural data and information available in the literature, we hypothesized that MsMutT1 could hydrolyze diphosphoinositol pentakisphosphate (PP-IP5 or IP7) to inositol hexakisphosphate (IP6), a reaction significant in many signaling processes. However, the individual domains of MsMutT1 do not hydrolyze IP7. In this thesis, we report three independent crystal structures of the complex between MsMutT1 and IP6. We note that the Nudix domain of MsMutT1 is the active site for the hydrolysis of IP7. However, the presence of the C-terminal domain is necessary for either recognition or efficient catalysis of IP7. This work is presented as an appendix in this thesis. | en_US |
