Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.Class switch recombination (CSR) is a DNA recombinational event that occurs in germinal-center B cells (6,14,39). By undergoing class switch recombination, mature B cells can switch their immunoglobulin heavy-chain constant regions from (which encodes the IgM subtype) to other heavy-chain isotypes (␥ for IgG, ␣ for IgA, and ⑀ for IgE, respectively). Class switch recombination is mediated by large (2-to 12-kb) repetitive regions, called switch regions, upstream of each coding constant-region exon (14). Recombination is thought to occur through a DNA double-strand break (DSB) intermediate (25). CSR generates DSBs that can be detected by ligationmediated PCR (4, 30, 36); moreover, the deleted intervening DNA between the two breaks forms a circular molecule (17,18,22,35), which is also detectable in class-switching B cells. The joining of the two chromosomal ends of the DSB is thought to be mediated by the nonhomologous DNA endjoining pathway (3,6,20,21), although dependence on some components is not certain (1).A key step in committing a certain switch region to recombination is transcription through that switch region from an upstream promoter (called an I exon promoter) (39). These transcripts are called germ line transcripts [to be distinguished from the transcript containing the V(D)J exon] or sterile transcripts, because they do not encode any protein (14, 39). The germ line transcripts are highly G rich, due to an asymmetry between a G-rich nontemplate and a C-rich template DNA strand of the switch region. The two switch regions involved in CSR do not share any extensive homology, and switch junctions do not reveal any consensus sequence motif. This type of DNA recombination event can be best described as regionally specific recombination (19,39).When switch regions are transcribed in vitro, the RNA transcripts stay bound to the DNA template, forming an RNA-DNA hybrid (9,...