The mechanism responsible for immunoglobulin class switch recombination is unknown. Previous work has shown that class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the gamma3 or the gamma2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the class switch recombination mechanism

Activation-induced deaminase (AID) is required for both immunoglobulin class switch recombination and somatic hypermutation. AID is known to deaminate cytidines in single-stranded DNA, but the relationship of this step to the class switch or somatic hypermutation processes is not entirely clear. We have studied the activity of a recombinant form of the mouse AID protein that was purified from a baculovirus expression system. We find that the length of the single-stranded DNA target is critical to the action of AID at the Cs positioned anywhere along the length of the DNA. The DNA sequence surrounding a given C influences AID deamination efficiency. AID preferentially deaminates Cs in the WRC motif, and additionally has a small but consistent preference for purine at the position after the WRC, thereby favoring WRCr (the lowercase r corresponds to the smaller impact on activity).

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R-loops have been described in vivo at the immunoglobulin class switch sequences and at prokaryotic and mitochondrial origins of replication. However, the biochemical mechanism and determinants of R-loop formation are unclear. We find that R-loop formation is nearly eliminated when RNase T 1 is added during transcription but not when it is added afterward. Hence, rather than forming simply as an extension of the RNA-DNA hybrid of normal transcription, the RNA must exit the RNA polymerase and compete with the nontemplate DNA strand for an R-loop to form. R-loops persist even when transcription is done in Li ؉ or Cs ؉ , which do not support G-quartet formation. Hence, R-loop formation does not rely on G-quartet formation. R-loop formation efficiency decreases as the number of switch repeats is decreased, although a very low level of R-loop formation occurs at even one 49-bp switch repeat. R-loop formation decreases sharply as G clustering is reduced, even when G density is kept constant. The critical level for R-loop formation is approximately the same point to which evolution drove the G clustering and G density on the nontemplate strand of mammalian switch regions. This provides an independent basis for concluding that the primary function of G clustering, in the context of high G density, is R-loop formation.R-loops are nucleic acid structures in which an RNA strand displaces one strand of DNA for a limited length in an otherwise duplex DNA molecule. R-loops were named by analogy to D-loops, which is where all three strands are DNA. R-loops form in vivo at sequences that generate a G-rich transcript at the prokaryotic origins of replication (20), mitochondrial origins of replication (18), and mammalian immunoglobulin (Ig) class switch sequences (reviewed in reference 45).