Mechanism of R-loop formation at immunoglobulin class switch sequences.
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.
Roy
Deepankar
D
USC Norris Comprehensive Cancer Ctr., Rm. 5428, 1441 Eastlake Ave., MC9176, Los Angeles, CA 90089-9176, USA.
Yu
Kefei
K
Lieber
Michael R
MR
D000818:Animals; D001483:Base Sequence; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D007136:Immunoglobulins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D006163:Ribonuclease T1 / Q000378:metabolism
D007136:Immunoglobulins; D006163:Ribonuclease T1