RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination

Abstract

Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination. We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions. In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments. We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels. These altered mobilities are resistant to RNase A but sensitive to RNase H. Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA. Importantly, complex formation only occurs upon transcription in the physiologic orientation. Reaban and Griffin [1990 Nature, 348, 342-344] found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment. Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand. These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination.