title: Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination.
abstract: During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR.
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Department of Biology, Swiss Federal Institute of Technology (ETH) Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bregenhorn
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Lia
authors.initials: L
authors.lastname: Kallenberger
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Mariela
authors.initials: M
authors.lastname: Artola-Borán
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland University of Copenhagen, Faculty of Health Sciences Center for Healthy Aging, Department of Neuroscience and Pharmacology, Blegdamsvej 3b, DK-2200 Copenhagen N, Denmark.
authors.forename: Javier
authors.initials: J
authors.lastname: Peña-Diaz
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Department of Biology, Swiss Federal Institute of Technology (ETH) Winterthurerstrasse 190, CH-8057 Zurich, Switzerland jiricny@imcr.uzh.ch.
authors.forename: Josef
authors.initials: J
authors.lastname: Jiricny
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D003564:Cytidine Deaminase / Q000235:genetics / Q000276:immunology; D003596:Cytosine / Q000276:immunology / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000276:immunology; D053903:DNA Breaks, Double-Stranded; D053843:DNA Mismatch Repair / Q000276:immunology*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D015398:Signal Transduction; D014498:Uracil / Q000276:immunology / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000276:immunology; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D007127:Immunoglobulin Constant Regions; D014498:Uracil; D003596:Cytosine; D004247:DNA; D051981:Uracil-DNA Glycosidase; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase