Transcription-dependent R-loop formation at mammalian class switch sequences

Immunoglobulin class switching is mediated by recombination between snitch sequences located immediately upstream of the immunoglobulin constant heavy chain genes. Targeting of recombination to particular switch sequences is associated temporally with transcription through these regions. We recently have provided evidence for inducible and stable RNA-DNA hybrid formation a t switch sequences in the mouse genome that are mechanistically important f or class snitching in vivo. Here, we define in vivo the precise configurati on of the DNA and RNA strands within this hybrid structure at the S mu, S g amma 3 and S gamma 2b mouse snitch sequences. We find that the G-rich (non- template) DNA strand of each switch sequence is hypersensitive to probes th roughout much of its length, while the C-rich (template) DNA strand is esse ntially resistant. These results demonstrate formation of an R-loop, whereb y the G-l ich RNA strand forms a stable heteroduplex with its C-rich DNA st rand counterpart, and the G-rich DNA strand exists primarily in a single-st randed state. We propose that the organized structure of the R-loop is esse ntial for targeting the class switch recombination machinery to these seque nces.