DNA Repair 11 (2012) 587–593 Contents lists available at SciVerse ScienceDirect DNA Repair jo u rn al hom epa ge: www.elsevier.com/locate/dnarepair Brief report Strikingly different properties of uracil-DNA glycosylases UNG2 and SMUG1 may explain divergent roles in processing of genomic uracil Berit Doseth, Cecilie Ekre, Geir Slupphaug, Hans E. Krokan, Bodil Kavli ∗ Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway a r t i c l e i n f o Article history: Received 14 December 2011 Received in revised form 13 March 2012 Accepted 13 March 2012 Available online 6 April 2012 Keywords: Cytosine deamination Uracil-DNA glycosylase Substrate specificity Activation-induced cytidine deaminase Hot-spot sequence a b s t r a c t Genomic uracil resulting from spontaneously deaminated cytosine generates mutagenic U:G mismatches that are usually corrected by error-free base excision repair (BER). However, in B-cells, activation-induced cytosine deaminase (AID) generates U:G mismatches in hot-spot sequences at Ig loci. These are subject to mutagenic processing during somatic hypermutation (SHM) and class switch recombination (CSR). Uracil N-glycosylases UNG2 and SMUG1 (single strand-selective monofunctional uracil-DNA glycosylase 1) initiate error-free BER in most DNA contexts, but UNG2 is also involved in mutagenic processing of AID-induced uracil during the antibody diversification process, the regulation of which is not understood. AID is strictly single strand-specific. Here we show that in the presence of Mg 2+ and monovalent salts, human and mouse SMUG1 are essentially double strand-specific, whereas UNG2 efficiently removes uracil from both single and double stranded DNA under all tested conditions. Furthermore, SMUG1 and UNG2 display widely different sequence preferences. Interestingly, uracil in a hot-spot sequence for AID is 200-fold more efficiently removed from single stranded DNA by UNG2 than by SMUG1. This may explain why SMUG1, which is not excluded from Ig loci, is unable to replace UNG2 in antibody diversification. We suggest a model for mutagenic processing in which replication protein A (RPA) recruits UNG2 to sites of deamination and keeps DNA in a single stranded conformation, thus avoiding error-free BER of the deaminated cytosine. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Spontaneous deamination of cytosine to uracil in DNA gen- erates mutagenic U:G mispairs at a rate of ∼70–200 events per human genome per day [1]. However, in follicular B cells activation-induced cytidine deaminase (AID) deaminates cytosines in immunoglobulin loci in antigen-driven antibody diversification, in which AID and DNA repair protein UNG2 serve as mutator proteins [2], unlike the role of UNG2 in error-free BER [1]. Further- more, off-target deamination by AID at other gene loci is linked to oncogenic mutations and translocations in B-cell lymphoma [3]. Mammalian cells express four nuclear UDGs. These are UNG2, Abbreviations: AID, activation-induced cytidine deaminase; AP, apurinic/apyrimidinic; APE1, AP-endonuclease; BER, base excision repair; CSR, class switch recombination; HIGM, hyper-IgM; IP, immunoprecipitation; MBD4, methyl-binding domain protein 4; PCNA, proliferating cell nuclear antigen; RPA, replication protein A; SHM, somatic hypermutation; SMUG1, single-strand-selective monofunctional uracil-DNA glycosylase 1; TDG, thymine-DNA glycosylase; UDG, uracil-DNA glycosylases; UNG, uracil N-glycosylase. ∗ Corresponding author at: Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, PO-Box 8905, N-7491 Trondheim, Norway. Tel.: +47 72573221; fax: +47 72 57 64 00. E-mail address: bodil.kavli@ntnu.no (B. Kavli). SMUG1 (single strand-selective monofunctional UDG) [4], TDG and MBD4. TDG and MBD4 remove uracil only from U:G mismatches and preferentially in CpG contexts [5]. In spite of the name, SMUG1 also removes uracil from dsDNA [6–9] and UNG2 and SMUG1 are unique by excising uracil from both single stranded (ss) and double stranded (ds) DNA in U:A as well as U:G contexts [7]. In human cells, UNG2 is the major enzyme for removal of uracil from both U:A, U:G and ssDNA [7,10,11]. However, in mice SMUG1 is important for repair of U:G mismatches [6]. The discrepancy between results for human and mouse UNG2 is most likely due to species differences [11]. Importantly, UNG2 is essential for SHM and CSR and SMUG1 can- not substitute for UNG2-deficiency in mouse and man [12,13]. AID deaminates cytosine in single stranded DNA during active transcription [14] and displays sequence preferences with WRCY (W = A/T, R = purine, Y = pyrimidine) being a hotspot [15–18]. How- ever, sequence preferences of nuclear UNG2 and SMUG1 in dsDNA and ssDNA contexts have not previously been investigated in any detail. Here we report that the properties of UNG2 are favorable in mutagenic processing of uracil-residues generated by AID in Ig loci, while those of SMUG1 are not. Thus, the divergent functions of UNG2 and SMUG1 are revealed already at the genomic uracil context level. 1568-7864/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2012.03.003