Role of a strictly conserved residue in N‐glycosylic bond cleavage by human Thymine DNA Glycosylase

Abstract

Thymine DNA glycosylase (TDG) promotes genomic integrity by removing thymine from mutagenic G·T mispairs arising from deamination of 5‐methylcytosine and follow‐on base excision repair enzymes restore a G·C pair. Previous studies suggested that Asn140, a strictly conserved active site residue is important, for base excision, yet its catalytic role had not been investigated rigorously. To further our understanding of the catalytic mechanism of TDG, here we determined the contribution of Asn140 to the substrate binding and chemical steps of the reaction. Isothermal titration calorimetry (ITC) experiments show that TDG‐N140A variant binds substrate analogs with the same tight affinity as wild‐type TDG, indicating Asn140 does not contribute to substrate binding. Single turnover kinetics experiments show that TDG‐N140A exhibits no detectable base excision activity for a G·T substrate, and its excision rate is vastly diminished (by ~104.4fold) for G·U, G·FU, and G·BrU substrates. Our findings indicate that Asn140 is essential for the chemical step, but does not contribute substantially to substrate binding. Thus N140A variant provides a useful platform for investigating the role of other residues in forming the reactive enzyme‐substrate complex. This work was supported by an NIH grant.