Formation of RNA-DNA hybrid, or R-loop, was studied in vitro by transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formation in cis was diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loop in trans with DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling. 1. Introduction The DNA-dependent RNA polymerase separates product RNA from its template DNA, thereby making the RNA available to ensuing cellular processes [1, 2]. The polymerase possesses structural elements for this separation and the RNA transcript is extruded through a hole of the polymerase molecule [3–6]. Despite this “separator” function of the polymerase, some transcripts have been known to anomalously form an RNA:DNA hybrid, or an R-loop, with their template, and R-loops have been implicated in a number of biological processes [7]. One classical example is the colE1 replication origin, where the RNA in the R-loop serves as the primer of DNA replication [8]. A more recent example is the FLOWERING LOCUS C of Arabidopsis thaliana, where an R-loop at the promoter of the COOLER gene represses the transcription of the gene [9]. In addition to the “separator” function of the RNA polymerase, cellular processes ensuing transcription, such as splicing and RNA export, also serve to sequester RNA from the template DNA. Compromising such a function has been shown to result in hyperrecombination [7, 10]. R-loops have also been observed at G-rich repetitive sequences in immunoglobulin class-switch regions, although R-loop per se is not considered to trigger class-switching [11–16]. R-loop formation at a class-switch region was first shown for the murine region [11]. Transcription of supercoiled plasmid DNA containing a 2.3？kb fragment of the region resulted in relaxation of the DNA, and the relaxation was reversed by RNase H treatment, which indicated R-loop formation. The relaxation was dependent on the direction of transcription. Radiolabeling experiments showed that the R-loop formed also when the template was linearized and that the RNA content in the R-loop was solely A and G [12]. The latter result, together with the length of the RNase A-resistant RNA bound to the DNA, indicated that the R-loop formed at the 28 tandem repeats of AGGAG in the region. Despite the simple repetitive nature of the sequence, the exact mechanism of the R-loop formation
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