Downstream boundary of chromosomal R-loops at murine switch regions: Implications for the mechanism of class switch recombination
- *Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Chemistry, and
- †Departments of Urology and Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089-9176
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Edited by Frederick W. Alt, Harvard Medical School, Boston, MA, and approved February 6, 2006 (received for review August 1, 2005)
Abstract
R-loops form at Sγ3 and Sγ2b 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 Sγ3 and Sγ2b 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 Sγ3 and the Sγ2b 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.
Footnotes
- ‡To whom correspondence should be addressed at: University of Southern California Norris Comprehensive Cancer Center, Room 5428, Los Angeles, CA 90033. E-mail: lieber{at}usc.edu
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Author contributions: F.-T.H., K.Y., C.-L.H., and M.R.L. designed research; F.-T.H. and K.Y. performed research; C.-L.H. contributed new reagents/analytic tools; F.-T.H., K.Y., C.-L.H., and M.R.L. analyzed data; and F.-T.H. and M.R.L. wrote the paper.
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Conflict of interest statement: No conflicts declared.
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This paper was submitted directly (Track II) to the PNAS office.
- Abbreviation:
- CSR,
- class switch recombination.
- © 2006 by The National Academy of Sciences of the USA
