Mutation Research 736 (2012) 25–32 Contents lists available at SciVerse ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis jo ur n al hom ep a ge: www.elsevier.com/locate/molmut C om mun i ty a ddress: www.elsevier.com/locate/mutres Progress in high-throughput assays of MGMT and APE1 activities in cell extracts Panagiotis Georgiadis ∗ , Nektaria Polychronaki, Soterios A. Kyrtopoulos National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, 48 Vassileos Constantinou Ave., Athens 11635, Greece a r t i c l e i n f o Article history: Received 17 January 2012 Received in revised form 26 April 2012 Accepted 9 May 2012 Available online 17 May 2012 Keywords: MGMT O 6 -methylguanine APE1 BER pathway Phenotypic assay Molecular epidemiology a b s t r a c t DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O 6 -methylguanine-DNA-methyltransferase (MGMT), which repairs the O 6 -alkylguanine-type of adducts induced in DNA by alkylating genotoxins; and (2) apurinic/apyrimidinic endonuclease 1 (APE 1), which participates in base excision repair (BER) by causing a rate-limiting DNA strand cleavage 5 ′ to the abasic sites. The MGMT assay makes use of the fact that: (a) the enzyme works by irreversibly transferring the alkyl group from the O 6 position of guanine to a cystein residue in its active site and thereby becomes inactivated and (b) that the free base O 6 -benzylguanine (BG) is a very good substrate for MGMT. In the new assay, cell extracts are incubated with BG tagged with biotin and the resulting MGMT-BG- biotin complex is immobilized on anti-MGMT-coated microtiter plates, followed by quantitation using streptavidin-conjugated alkaline phosphatase and a chemiluminescence-producing substrate. A one-step/one-tube phenotypic assay for APE1 activity has been developed based on the use of a flu- orescent molecular beacon (partially self-complementary oligonucleotide with a hairpin-loop structure carrying a fluorophore and a quencher at each end). It also contains a single tetrahydrofuran residue (THF) which is recognized and cleaved by APE1, and the subsequently formed single-stranded oligomer becomes a fluorescence signal emitter. Both assays are highly sensitive, require very small amounts of protein extracts, are relatively inexpensive and can be easily automated. They have been extensively validated and are being used in the context of large-scale molecular epidemiology studies. © 2012 Elsevier B.V. All rights reserved. 1. Introduction DNA repair is an important parameter contributing to individual susceptibility to the effects of genotoxic carcinogens. The impact of variations in DNA repair ability on individual cancer risk has been demonstrated in population studies of genetic variations at DNA repair-related loci as well as studies based on measurement of repair activity using phenotypic assays (for recent reviews see Refs. [1–4]). Phenotypic DNA repair assays have an advantage compared to studies based on genetic variation, in that they directly reflect the activity of the repair proteins or multi-protein complexes in question, thus accounting for variations in the level of expression as well as in the catalytic efficacy. On the other hand, they tend to be technically complex and time-consuming, in contrast to genotyp- ing assays, a disadvantage which assumes particular significance in view of the trend towards the use of large population sizes (in the ∗ Corresponding author. Tel.: +30 210 7273733; fax: +30 210 7273677. E-mail address: panosg@eie.gr (P. Georgiadis). hundreds or even thousands) in molecular epidemiology studies. Thus there is an increasing need for phenotypic repair assays which are relatively simple to conduct, efficient and of low cost. Here we present the development and validation of two phenotypic DNA repair assays which respond to this demand, concerning the repair proteins O 6 -methylguanine-DNA methyltransferase (MGMT) and apurinic endonuclease 1 (APE1). MGMT is a protein which repairs O 6 -methylguanine (O6- meG), a cytotoxic, mutagenic and carcinogenic lesion generated by DNA by methylating agents [5]. If not repaired, these lesions induce cell death by activating apoptotic pathways [6]. Methylat- ing agents include chemicals of environmental relevance (dietary or endogenously formed) such as N-nitroso compounds (e.g. N-nitrosodimethylamine), as well as certain drugs (e.g. temo- zolomide, procarbazine and dacarbazine) widely used in cancer chemotherapy. Quantification of MGMT is an important biomarker of susceptibility, since human exposure to carcinogenic N-nitroso compounds is ubiquitous [7] and accumulated evidence suggests an influence of MGMT polymorphisms on cancer risk [8]. Interest in the measurement of MGMT activity in human samples also arises 0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2012.05.002