TY - JOUR
T1 - 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
AU - Maul, Robert W.
AU - Chon, Hyongi
AU - Sakhuja, Kiran
AU - Cerritelli, Susana M.
AU - Gugliotti, Lina A.
AU - Gearhart, Patricia J.
AU - Crouch, Robert J.
N1 - Publisher Copyright:
© 2017
PY - 2017/10/27
Y1 - 2017/10/27
N2 - 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.
AB - 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.
KW - R-loops
KW - RNase H1
KW - class switch recombination
KW - somatic hypermutation
KW - transgenic mouse
UR - http://www.scopus.com/inward/record.url?scp=85009485526&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85009485526&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2016.12.020
DO - 10.1016/j.jmb.2016.12.020
M3 - Article
C2 - 28065739
AN - SCOPUS:85009485526
SN - 0022-2836
VL - 429
SP - 3255
EP - 3263
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 21
ER -