Downstream boundary of chromosomal R-loops at murine switch regions: Implications for the mechanism of class switch recombination

摘要

R-loops form at S gamma 3 and S gamma 2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine S gamma 3 and S gamma 2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the S gamma 3 and the S gamma 2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination.