DNA Repair 11 (2012) 559–569 Contents lists available at SciVerse ScienceDirect DNA Repair jo u rn al hom epa ge: www.elsevier.com/locate/dnarepair The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA Kathrin Torseth a , Berit Doseth a , Lars Hagen a , Camilla Olaisen a , Nina-Beate Liabakk a , Heidi Græsmann a,b , Anne Durandy c , Marit Otterlei a , Hans E. Krokan a , Bodil Kavli a,∗∗ , Geir Slupphaug a,∗ a Department of Cancer Research and Molecular Medicine, Faculty of Medicine, the FUGE Proteomics Node, Norwegian University of Science and Technology, N-7006 Trondheim, Norway b Department of Biomedical Science, The Faculty of Technology (AFT), Sør-Trøndelag University College (HiST), N-7004 Trondheim, Norway c INSERM, U768, Hôpital Necker-Enfants Malades, Université Paris Descartes, Faculté de Médecine Paris V-René Descartes, Paris F-75005, France a r t i c l e i n f o Article history: Received 17 November 2011 Received in revised form 28 March 2012 Accepted 28 March 2012 Available online 20 April 2012 Keywords: Uracil-DNA glycosylase UNG2 variant Base excision repair RPA PCNA a b s t r a c t In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruit- ment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells. © 2012 Elsevier B.V. All rights reserved. Abbreviations: AID, activation-induced cytidine deaminase; AP, apurinic/apyrimidinic; BER, base excision repair; CSR, class-switch recombination; ESI-MS, electrospray ionization mass spectrometry; HIGM syndrome, hyper-IgM syndrome; LCL, lymphoblastoid cell line; MALDI-TOF MS, matrix assisted laser desorption ionization time of flight mass spectrometry; PCNA, proliferating cell nuclear antigen; RPA, replication protein A; SHM, somatic hypermutation; UDG, uracil-DNA glycosylase; UNG, UDG of the UNG family; UNG1, mitochondrial UNG; UNG2, nuclear UNG. ∗ Corresponding author. Tel.: +47 72573076; fax: +47 72576400. ∗∗ Corresponding author. Tel.: +47 72573221; fax: +47 72576400. E-mail addresses: kathrin.torseth@ntnu.no (K. Torseth), berit.doseth@ntnu.no (B. Doseth), lars.hagen@ntnu.no (L. Hagen), camilla.olaisen@ntnu.no (C. Olaisen), nina.beate.liabakk@ntnu.no (N.-B. Liabakk), heidi.sterten@hist.no (H. Græsmann), anne.durandy@inserm.fr (A. Durandy), marit.otterlei@ntnu.no (M. Otterlei), hans.krokan@ntnu.no (H.E. Krokan), bodil.kavli@ntnu.no (B. Kavli), geir.slupphaug@ntnu.no (G. Slupphaug). 1. Introduction The RNA base uracil is not a normal component of DNA, but nevertheless occurs in small amounts in the genomes of living organisms due to deamination of cytosine and misincorporation of dUMP during replication. Uracil generated by deamination of cytosine is 100% miscoding, and C:G to T:A transition mutations are introduced if these lesions are not repaired before replication. In contrast, replicative incorporation of dUMP generates U:A base pairs that are not directly miscoding, but may produce cytotoxic and mutagenic AP site intermediates during repair [1]. In most cells uracil-induced mutagenesis is counteracted by the base-excision repair (BER) pathway, in which a uracil-DNA glyco- sylase excises the uracil, followed by cleavage of the abasic site, replacement of the deoxyribose with dCMP or dTMP, and finally, ligation to restore the original DNA sequence [1]. Misincorporated 1568-7864/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2012.03.006