Structure-function and evolution of a unique uracil DNA glycosylase, UdgX, from Mycobacterium smegmatis

Abstract

Mycobacterium smegmatis, a soil bacterium, faces multiple challenges in its environment, including exposure to reactive oxygen species (ROS), reactive nitrogen intermediates (RNI), radiation, and various other chemical stressors. These environmental stresses inflict significant damage on the bacterium's cellular components, with DNA and its precursors being particularly significant. The high G+C content in mycobacteria makes them more susceptible to such harm, requiring development of protective mechanisms. Among these mechanisms, a crucial process involves repairing uracil residues in DNA, resulting from cytosine deamination or their direct incorporation from dUTP, during DNA synthesis. Playing a vital role in this repair pathway are the enzymes, uracil DNA glycosylases (UDGs), which catalyze excision and repair of uracils through the base excision repair (BER) pathway. M. smegmatis possesses three UDGs: Ung, UdgB, and the distinctive UdgX. Notably, UdgX stands apart from the other two UDGs by its ability to form covalent complexes with uracil-containing DNA, indicating a unique role for the UdgX protein in the bacterium. The primary objective of this study is to explore the structure, mechanism, evolution, and function of the distinctive UDG, MsmUdgX (MSMEG_0265). By gaining insights into these protective mechanisms, we aim to better comprehend how mycobacteria adapt to their challenging environment. Previous investigations on MsmUdgX have revealed its remarkable specificity in recognizing uracils within DNA strands, leading to the formation of stable complexes. An intriguing finding was the conversion of UdgX into a conventional UDG, lacking the property of tight complex formation with DNA upon mutation of H109 to many other amino acids. The studies have led to a more comprehensive understanding of the mechanism of action, evolution, and function of UdgX from M. smegmatis. 1. Deciphering the nature of UdgX-uracil DNA complex, and the evolution of UdgX family proteins This chapter presents extensive investigations into the mechanism and function of MsmUdgX, a unique uracil DNA glycosylase found in M. smegmatis. Previous studies demonstrated specific recognition of uracils in both single and double-stranded DNA by MsmUdgX to form a stable complex. However, further understanding of the chemical nature of the complex formation and the conversion of UdgX (H109S) into a conventional UDG required additional research. The current study aimed to comprehensively unravel the biochemical and structural basis of UdgX function. Notably, MsmUdgX is the first reported uracil DNA glycosylase that forms a covalent complex with uracil-containing DNA concurrently with the excision of uracil, making this mechanism of uracil excision a primary area of interest for investigation. The research explored the interactions of MsmUdgX with DNA substrates and revealed that uracil is excised before UdgX forms a covalent complex with the DNA. Investigation into the residues involved in these interactions led to the identification of H109, which forms a covalent bond with the AP-DNA concurrently with the cleavage of the sugar-uracil glycosidic bond. Mutants of H109 exhibited conventional UDG activity with multiple turnovers, while the wild-type UdgX displayed a single turnover reaction, resembling a suicide enzyme in vitro. Crystal structures of the UdgX-DNA and UdgX-uracil complexes provided crucial insights into the residues responsible for UdgX-DNA interactions. Bioinformatics analysis identified signature residues distinguishing MsmUdgX from the family4 UDGs. Moreover, coevolution analysis indicated that the R-loop residues evolved with specific changes in family4 UDGs to form UdgX. Additionally, the study uncovered the role of Q53, a highly conserved residue in the UdgX active site. Q53 appears to dampen uracil excision activity and contributes to shaping the R-loop structure. These findings shed light on the unique characteristics and mechanisms of action of MsmUdgX, contributing to a deeper understanding of DNA repair processes in M. smegmatis. 2. Unravelling the unique mechanism of action of MsmUdgX This study focuses on the unique mechanism of action of UdgX, a novel uracil DNA glycosylase from M. smegmatis. Unlike conventional UDGs that excise uracil from DNA with turnover activity, UdgX excises uracil by cleavage of the N-glycosidic bond to form a covalent bond with the C1' position of the abasic deoxyribose sugar and the NE2 position of the crucial H109 residue. Structural analyses reveal that UdgX is closest to family4 UDGs but differs in its altered motif A (51GEQPG55) and the presence of the R-loop (105KRRIH109). Additionally, the study uncovered the role of E52, a highly conserved residue in the UdgX active site. E52 forms a catalytic dyad with H109, enhancing the nucleophilic attack by H109 on the target deoxyribose. Furthermore, the study highlights the importance of residue R184 in DNA binding, as evidenced by the R184A mutation, which results in altered Km and reduced Kcat/Km values for the mutant UdgX. These findings not only solidify the understanding of the catalytic mechanism of UdgX but also offer insights into its evolutionary relationship with family4 UDGs, suggesting an adaptation to perform its specialized role. This knowledge broadens the potential applications of engineered UdgX proteins in DNA technologies, expanding their ability to act on various DNA bases and enhancing their utility in diverse research and biotechnological contexts. 3. Preliminary biochemical and bioinformatics studies on the possible role of UdgX in M. smegmatis This study was aimed to uncover the mysterious role of UdgX in M. smegmatis, particularly its unique ability to form a covalent complex with the apDNA concurrently with uracil excision. In vivo experiments were conducted under diverse growth conditions to understand the physiological significance of UdgX. The investigation revealed significant variations in UdgX mRNA and protein levels during different growth/stress conditions, suggesting its involvement in DNA damage response. Detecting native UdgX protein levels was challenging but cloning it under a multicopy plasmid with its native promoter facilitated the analyses. The pull-down experiments identified potential interacting partners, including DnaK, MSMEG_0523, Endonuclease VIII, UvrB, and other proteins. However, further investigations are needed to fully confirm and understand the nature and significance of these interactions. The study speculated that the UdgX-DNA complex might interfere with DNA transactions, and the fate of the UdgX-DNA complex may involve protease-mediated degradation. Additionally, the presence of a potential pupylation site in UdgX suggested regulatory implications. Overall, this research sheds light on the role of UdgX in DNA repair and stress response. However, further studies are required to fully comprehend the identified interactions and the ultimate destiny of the UdgX-DNA complex.