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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Characterization of R loops in Class switch recombination

Recurrent DNA translocations characterize blood cell cancers, and are frequently products of aberrant repair of programmed DNA double-strand breaks (DSBs). Defining the precise molecular mechanisms governing DSB generation and repair is critical to revealing how lymphoid cancers arise. The majority of cancers involving antibody-producing B cells arise during class switch recombination (CSR), a programmed DNA repair event at the immunoglobulin heavy chain (IgH) locus. CSR is initiated by DSBs generated by the enzyme AID, whose recruitment to IgH requires transcription. AID-dependent DSB formation strongly correlates with the appearance of R loops—three-stranded nucleotide structures where newly transcribed RNA re-anneals to template DNA. Though R loops were observed at the IgH locus over 20 years ago, their role in CSR remains undefined. To investigate the role R loops play in CSR, we generated mice deficient for two enzymes promoting R loop dissolution, the helicase Senataxin (SETX-/- ) and the nuclease RNase H2 (RNH2B f/f). We find that B cells from SETX-/- RNH2B f/f mice have increased R loop formation, and over 10% of cells accumulate unrepaired DNA breaks and translocations at IgH. In contrast, WT and single mutants show modest IgH instability and limited increases in R loop formation. All 4 genotypes are proficient for CSR, thus DSB repair is largely intact. These results show that Setnataxin and RNase H2 act independently to promote genome stability and suppress aberrant DNA repair during CSR. Sequence analysis revealed an increased frequency of insertions and deletions at class switch junctions in double-deficient cells, a mutational signature associated with increased repair via alternative end-joining. We propose that Senataxin and Rnase H2 promote classical NHEJ by removing R loops during CSR.

This research has been supported by an NCI Career Transition Award, K22CA188106

Jacqueline Barlow
Hongchang Zhao

Department of Microbiology and Molecular Genetics
University of California Davis
Davis, CA 955616

jhbarlow@ucdavis.edu