DNA Breaks in Ig V Regions Are Predominantly Single Stranded and Are Generated by UNG and MSH6 DNA Repair Pathways

Abstract

Antibody diversity is initiated by activation-induced deaminase (AID), which deaminates cytosine to uracil in DNA. Uracils in the Ig gene loci can be recognized by uracil DNA glycosylase (UNG) or mutS homologs 2 and 6 (MSH2–MSH6) proteins, and then processed into DNA breaks. Breaks in switch regions of the H chain locus cause isotype switching and have been extensively characterized as staggered and blunt double-strand breaks. However, breaks in V regions that arise during somatic hypermutation are poorly understood. In this study, we characterize AID-dependent break formation in JH introns from mouse germinal center B cells. We used a ligation-mediated PCR assay to detect single-strand breaks and double-strand breaks that were either staggered or blunt. In contrast to switch regions, V regions contained predominantly single-strand breaks, which peaked 10 d after immunization. We then examined the pathways used to generate these breaks in UNG- and MSH6-deficient mice. Surprisingly, both DNA repair pathways contributed substantially to break formation, and in the absence of both UNG and MSH6, the frequency of breaks was severely reduced. When the breaks were sequenced and mapped, they were widely distributed over a 1000-bp intron region downstream of JH3 and JH4 exons and were unexpectedly located at all 4 nt. These data suggest that during DNA repair, nicks are generated at distal sites from the original deaminated cytosine, and these repair intermediates could generate both faithful and mutagenic repair. During mutagenesis, single-strand breaks would allow entry for low-fidelity DNA polymerases to generate somatic hypermutation.