Biochemical Analyses of UDGX-A Crosslinking Uracil-Dna Glycosylase

DNA base damage is common due to exposure to various endogenous and exogenous factors. To repair the base lesions, such as uracil from cytosine deamination, enzymes from the uracil-DNA glycosylase (UDG) superfamily are critical, which can recognize the damaged base and initiate the base excision repair (BER) pathway. There used to be six families of proteins identified in the UDG superfamily until a new member, UDGX, was found in Mycobacterium smegmatis, which is a unique DNA-crosslinking UDG. In this dissertation work, a series of biochemical analyses of the newly found UDGX are performed, including the analyses of structures, functions, catalytic mechanism, distribution in species and even potential evolutionary trends. Chapter 1 is a general introduction of common DNA base damage and enzymes from different families in the UDG superfamily. Chapter 2 presents the structural and functional analyses of UDGX by mutational and kinetics studies. It possesses multiple inter-motif and intra-motif interactions of residues in the structure of UDGX, which results in the coupling of uracil excision and DNA crosslinking functions of UDGX. Based on the understanding of the structural and functional coupling, a catalytic mechanism of UDGX on uracil-DNA substrates is also proposed. In Chapter 3, the DNA crosslinking site, the residue H109 of UDGX is investigated. By mutational and kinetics analyses, the function coupling of UDGX is further confirmed; additionally, a unique mutant, H109E was also found possessing DNA crosslinking capability via an ester bond with the abasic site instead. In Chapter 4, the DNA crosslinking activities of six putative UDGX homologs from different species were screened. It has been found that there are probably some other interactions of residues outside motifs essential to the activities of UDGX. For example, we found a salt bridge between R158 and D200 that is critical when we studied two UDGX homologs which have extremely similar sequences to the Msm UDGX. In conclusion, with these biochemical analyses of UDGX, we have achieved a better understanding of this unique bifunctional DNA-crosslinking UDG, which expands the biological significance and application of enzymes in UDG superfamily