title: MD simulation and experimental evidence for Mg²+ binding at the B site in human AP endonuclease 1.
abstract: Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.
authors.affiliation: Internal Medicine-Endocrinology Department, University of Texas Medical Branch, Galveston, TX 77555-1060, USA. nuoezgue@utmb.edu
authors.forename: Numan
authors.initials: N
authors.lastname: Oezguen
authors.forename: Anil K
authors.initials: AK
authors.lastname: Mantha
authors.forename: Tadahide
authors.initials: T
authors.lastname: Izumi
authors.forename: Catherine H
authors.initials: CH
authors.lastname: Schein
authors.forename: Sankar
authors.initials: S
authors.lastname: Mitra
authors.forename: Werner
authors.initials: W
authors.lastname: Braun
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