title: R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching.
abstract: R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch μ region were depleted by 70% in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells. However, somatic hypermutation was increased specifically on the transcribed strand from μ-γ joins, indicating that R-loops limit activation-induced (cytosine) deaminase access to the transcribed DNA strand. Our data suggest that, in the normal G+C-rich context of mammalian class switch recombination regions, R-loops are obligatory intermediates. Processing of the R-loops is needed to remove RNA allowing activation-induced (cytosine) deaminase to promote somatic hypermutation on both DNA strands to generate double-strand DNA breaks for efficient class switch recombination. One of the two cellular RNases H may assist in this process.
authors.affiliation: Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Maul
authors.affiliation: Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Hyongi
authors.initials: H
authors.lastname: Chon
authors.affiliation: Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Sakhuja
authors.affiliation: Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Susana M
authors.initials: SM
authors.lastname: Cerritelli
authors.affiliation: Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Lina A
authors.initials: LA
authors.lastname: Gugliotti
authors.affiliation: Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA. Electronic address: gearhartp@mail.nih.gov.
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Gearhart
authors.affiliation: Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. Electronic address: crouchr@helix.nih.gov.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Crouch
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D005260:Female; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D016914:Ribonuclease H / Q000502:physiology*; D027041:Somatic Hypermutation, Immunoglobulin; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D007132:Immunoglobulin Isotypes; D016914:Ribonuclease H; C078856:ribonuclease HI; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase
keywords: R-loops; RNase H1; class switch recombination; somatic hypermutation; transgenic mouse