DNA Repair 1 (2002) 507–516 Alkylation resistance of E. coli cells expressing different isoforms of human alkyladenine DNA glycosylase (hAAG) Kenneth Bonanno a,1 , Jennifer Wyrzykowski a , Wincha Chong a,2 , Zdenka Matijasevic b , Michael R. Volkert a,∗ a Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA b Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA Received 8 February 2002; accepted 3 April 2002 Abstract The alkyladenine DNA glycosylase (AAG) has been cloned from mouse and humans. AAG knock out mouse cells are sensitized to a variety of alkylating and cross-linking agents suggesting AAG is active on a variety of substrates. In humans, two isoforms have been characterized that are generated by alternative splicing and contain either exon 1a or 1b (hAAG1 or hAAG2). In this study, we examine the ability of the both known isoforms of human AAG (hAAG) to contribute to survival of Escherichia coli from treatments with simple alkylating agents and cross-linking alkylating agents. Our results show that hAAG is effective at repairing methyl lesions when expressed in E. coli, but is unable to afford increased resistance to alkylating agents producing larger alkyl lesions such as ethyl lesions or lesions produced by the cross-linking alkylating agents N,N ′ -bis-chloroethyl-N-nitrosourea (BCNU), N-(2-chloroethyl)-N-nitrosourea (CNU) or mitomycin C. In the case of CNU, expression of hAAG causes increased sensitivity rather than resistance, suggesting deleterious effects of hAAG activity. We also demonstrate that there are no apparent differences between the two isoforms of hAAG when recovery from damage produced by all alkylating agents is tested. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Alkylation resistance; E. coli cells; Human alkyladenine DNA glycosylase 1. Introduction The human alkyladenine DNA glycosylase gene (hAAG) was identified by its ability to complement methyl methane sulfonate (MMS) sensitivity of bac- ∗ Corresponding author. Tel.: +1-508-856-2314; fax: +1-508-856-5920. E-mail address: michael.volkert@umassmed.edu (M.R. Volkert). 1 Present address: Millennium Pharmaceuticals, Cambridge, MA 02139, USA. 2 Present address: Transitional Residency Program, University of Hawaii Medical School, Honolulu, HI 96813-2427, USA. terial mutants lacking their own AAG (AlkA and TagA) [1–3]. Studies with mouse embryonic stem (ES) cells indicate that mutation of the mouse AAG (mAAG) gene causes sensitivity not only to the methy- lating agents MMS and MeOSO 2 (CH 2 ) 2 ′ -lexitropsin, but also to the DNA cross-linking agents N,N ′ -bis- chloroethyl-N-nitrosourea (BCNU) and mitomycin C; no effects on UV sensitivity are detected [4,5]. In contrast, studies by others have shown that mouse embryonic fibroblast (MEF) cells carrying the AAG knockout mutation are sensitized to the lethal and mutagenic effects of high doses of MMS, but not to 1568-7864/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S1568-7864(02)00051-4