Mechanism of R‐Loop formation at Immunoglobulin Class Switch sequences

Abstract

Received 12 July 2007/Returned for modification 21 August 2007/Accepted 9 October 2007 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 T1 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). In addition, R-loop formation occurs in vivo at some G-rich transcript locations that are distinctly high for mitotic recombination in Saccharomyces cerevisiae, and this high recombination rate is reduced upon overexpression of S. cerevisiae RNase H1 (14). When prokaryotes lack topoisomerase activity, R-loops can form at a wider variety of sequences, and the lethality associated with this can be remedied by overexpression of Escherichia coli RNase H1 (7). In an avian lymphoid cell line, lack of the ASF2/SF2 RNA-binding protein favors R-loop formation at G-rich transcript locations in the genome, and expression of human RNase H1 can abolish the R-loop (19).