Targeted hypermutation of immunoglobulin variable region genes occurs in B cells during an immune response1, and gives rise to families of related mutant antibodies which are then selected for their binding affinity to the immunizing antigen2. Somatic hypermutation predominantly generates point mutations, many of which occur at specific residues (hotspots)3. The reaction has been linked to transcription and requires the presence of immunoglobulin enhancers4,5,6, but replacement of the variable gene by heterologous sequences, or the variable region promoter by a heterologous promoter, does not interfere with the mutation process7,8. Here we show the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences. Generation of the DSBs is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations. Furthermore, the DSBs are cell-cycle restricted, being found almost exclusively in cells that have completed, or nearly completed, DNA replication. We propose a model for somatic hypermutation in which mutations are introduced into the DNA during repair of DSBs by homologous recombination. The finding of DSBs during somatic hypermutation may help to explain the chromosomal translocations found in some B-cell tumours.
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We thank T. Taylor and G. Tokmoulina for help with cell sorting; E. Hilton for special assistance with DNA sequencing; C. Arthur for her role in the creation of transgenic mice; and S. Fugmann and M. Diaz for many helpful discussions. We are very grateful to the following people for helpful comments on the manuscript: M. Diaz, S. Fugmann, J. Haber, D. Hesslein, M. Jasin, M. Nussenzweig and I. Villey. Oligonucleotide synthesis and DNA sequencing were performed by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. F.N.P. was supported by a postdoctoral fellowship from the Arthritis Foundation and D.G.S. is an associate investigator of the Howard Hughes Medical Institute.
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