title: The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA.
abstract: 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 recruitment 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.
authors.affiliation: Department of Cancer Research and Molecular Medicine, Faculty of Medicine, the FUGE Proteomics Node, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Torseth
authors.forename: Berit
authors.initials: B
authors.lastname: Doseth
authors.forename: Lars
authors.initials: L
authors.lastname: Hagen
authors.forename: Camilla
authors.initials: C
authors.lastname: Olaisen
authors.forename: Nina-Beate
authors.initials: NB
authors.lastname: Liabakk
authors.forename: Heidi
authors.initials: H
authors.lastname: Græsmann
authors.forename: Anne
authors.initials: A
authors.lastname: Durandy
authors.forename: Marit
authors.initials: M
authors.lastname: Otterlei
authors.forename: Hans E
authors.initials: HE
authors.lastname: Krokan
authors.forename: Bodil
authors.initials: B
authors.lastname: Kavli
authors.forename: Geir
authors.initials: G
authors.lastname: Slupphaug
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016196:S Phase / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; C516794:RPA2 protein, human; C492570:CCNO protein, human; D045647:DNA Glycosylases