Magnesium, Essential for Base Excision Repair Enzymes, Inhibits Substrate Binding of N-Methylpurine-DNA Glycosylase

Abstract

is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.Cellular DNA is continuously exposed to endogenous or exogenous chemical or physical agents that induce DNA lesions. DNA base damage threatens genomic stability and cellular viability. Multiple DNA repair pathways exist in all organisms, from bacteria to humans, to preserve the integrity of the genome (1). If not repaired, damaged bases could be mutagenic (2) and/or cause cell death by blocking DNA replication (3).In all organisms, repair of DNA-containing small adducts, as well as altered and abnormal bases, occurs primarily via the base excision repair (BER) 2 pathway, beginning with cleavage of the base by a DNA glycosylase (1, 2). Mechanistically, DNA glycosylases are categorized into two classes: mono-and bifunctional DNA glycosylases. Monofunctional DNA glycosylases, such as N-methylpurine-DNA glycosylase (MPG) and uracil-DNA glycosylase, use an activated water molecule as a nucleophile to generate an apurinic or apyrimidinic (AP) site in DNA. Bifunctional DNA glycosylases/AP lyases, such as NTH1 and OGG1, use an activated amino group (Lys) or imino group (Pro) as the nucleophile to create a Schiff base intermediate that coordinates base removal and subsequent strand incision (AP lyase) 3Ј to the AP site (4, 5). The mammalian MPG is known to excise at least 17 structurally diverse modified bases from DNA (6). These lesions include 3-alkylpurines, 7-alkylguanine, 1,N 6 -ethenoadenine (⑀A), N 2 ,3-ethenoguanine, and hypoxanthine (Hx), all of which are purine derivatives (7-12). Moreover, the base alterations are located in both the major and minor grooves of duplex DNA. Its orthologs in Escherichia coli (AlkA) and yeast (MAG) have an overlapping although not identical substrate range. Nonetheless mammalian MPG and E. coli AlkA do not share significant sequence similarity or structural homology (13,14), despite this functional similarity and the fact that 3-methyladenine is a preferred substrate for both. MPG excises ⑀A and Hx more efficiently than AlkA and MAG (11), but unlike AlkA, it cannot excise O 2 -alkylpyrimidines (15, 16) and oxidized bases such as 5-formyluracil and 5-hydroxymethyluracil (17) (22); however, the reduction was more pronounced for the AP-lyase activity. The Schiff base formation between hOGG1-and 8-oxoG-containing DNA was abrogated in the presence of Mg 2ϩ . These results suggest that hOGG1 operates mainly as a monofunctional glycosylase under physiologic concentrations of Mg 2ϩ (22). There

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

“…Notably, k pd is composed of k off values for both free base and the AP site-containing DNA. Moreover, the k ss consists of k pd and the effective rate constant (kЈ) for the process MPG ϩ ⑀A-DNA 3 MPG⅐AP site DNA⅐⑀A (26). Generally, for obtaining the definite value for product dissociation from burst kinetics, it is assumed that k pd is much slower than the kЈ, and thus, the contribution of the latter is ignored, and k ss becomes identical to k pd .…”

“…The products containing the AP sites were analyzed as described previously (26). Percent product formation was determined by quantifying the substrate and product band intensities using an imager (Chemigenius Bioimaging System, Frederick, MD) and software (GeneTool, Syngene Inc., San Diego, CA).…”

“…The oligonucleotides were purified on a sequencing gel. The ⑀A-or Hx-containing oligonucleotide was labeled at the 5Ј-end using T4 polynucleotide kinase and [␥-32 P]ATP and then annealed to complementary oligonucleotide to prepare 5Ј-32 P-labeled duplex oligonucleotide as described previously (26).…”

“…Next, we attempted to measure the k pd (26,39). For the reaction of WT hMPG with ⑀A-DNA, we previously found a k pd value of 0.00435 Ϯ 0.00057 s Ϫ1 (39).…”

“…We conducted single turnover kinetics with hMPG proteins to measure the k chem (Fig. 5A and see "Experimental Procedures" for definition) (26,39). Two different enzyme/substrate ratios for both WT and variant proteins provided similar k chem values, ensuring that the enzymatic reactions were following single turnover conditions.…”