Abstract:
: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.
journal_name
Mol Cell Bioljournal_title
Molecular and cellular biologyauthors
Roy D,Yu K,Lieber MRdoi
10.1128/MCB.01251-07subject
Has Abstractpub_date
2008-01-01 00:00:00pages
50-60issue
1eissn
0270-7306issn
1098-5549pii
MCB.01251-07journal_volume
28pub_type
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