Insights Into the Debated Lyase Mechanism of Bifunctional DNA Glycosylases From MD and QM/MM MD Simulations: The Case Study of DNA Oxidative Damage Repair by hOGG1

DNA damage induced by reactive oxygen species (ROS) can result in mutations that contribute to the development of human diseases such as cancer, neurological disorders, cardiovascular disease, and diabetes. Human 8-oxoguanine DNA glycosylase (hOGG1) is responsible for repairing the major DNA oxidative product, namely 8-oxoguanine (8oG). hOGG1 is a member of the bifunctional DNA glycosylase family, which cleaves both the glycosidic and phosphodiester bonds in damaged nucleotides as part of base excision repair (BER). While nucleotide deglycosylation to yield an enzyme-DNA imine crosslink has been well studied both experimentally and 2 computationally for several bifunctional glycosylases, relatively little is known about the subsequent and typically rate-limiting phosphodiester bond cleavage step. To gain the first atomic level details of the β-lyase pathway for a bifunctional glycosylase, the present study uses a combination of classical MD and QM/MM MD simulations to characterize the hOGG1 mechanism of action. Our simulations reveal that the cleaved 8oG glycosylation product rapidly leaves the active site, precluding the previously proposed product-assisted elimination and supporting the allosteric nature of 8oG activators. Although QM/MM MD calculations suggest a neutral crosslink prevents lyase activity, a pathway involving initial hydrolysis of a cationic crosslink followed by D268-catalyzed phosphate elimination is catalytically feasible and the first proposed mechanism to unify all existing experimental kinetic, mutagenic, and structural data. Furthermore, K249 is demonstrated to be a viable substitute for D268 in the elimination step, rationalizing reports that hOGG1 can accommodate D268 mutation. Our newly characterized mechanism of action can push forward the development of small molecule hOGG1 inhibitors and activators as disease therapeutics, while key mechanistic features may be generalizable to understand the function of other bifunctional glycosylases.