DNA Breaks in Hypermutating Immunoglobulin Genes: Evidence for a Break-and-Repair Pathway of Somatic Hypermutation

Corresponding author: Nancy Maizels, Departments of Immunology and Biochemistry, University of Washington Medical School, Rm. H474A HSB, 1959 NE Pacific St., Box 357650, Seattle, WA 98195-7650., maizels{at}u.washington.edu (E-mail)

Communicating editor: N. ARNHEIM

To test the hypothesis that immunoglobulin gene hypermutation in vivo employs a pathway in which DNA breaks are introduced and subsequently repaired to produce mutations, we have used a PCR-based assay to detect and identify single-strand DNA breaks in 1 genes of actively hypermutating primary murine germinal center B cells. We find that there is a two- to threefold excess of breaks in 1 genes of hypermutating B cells, relative to nonhypermutating B cells, and that 1.3% of germinal center B cells contain breaks in the 1 gene that are associated with hypermutation. Breaks were found in both top and bottom DNA strands and were localized to the region of 1 that actively hypermutates, but duplex breaks accounted for only a subset of breaks identified. Almost half of the breaks in hypermutating B cells occurred at hotspots, sites at which two or more independent breaks were identified. Breaksite hotspots were associated with characteristic sequence motifs: a pyrimidine-rich motif, either RCTYT or CCYC; and RGYW, a sequence motif associated with hypermutation hotspots. The sequence motifs identified at breaksite hotspots should inform the design of substrates for characterization of activities that participate in the hypermutation pathway.

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