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Molecular and Cellular Biology, January 2008, p. 50-60, Vol. 28, No. 1
0270-7306/08/$08.00+0     doi:10.1128/MCB.01251-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences{triangledown} ,{dagger}

Deepankar Roy, Kefei Yu,{ddagger} and Michael R. Lieber*

USC Norris Comprehensive Cancer Ctr., Rm. 5428, and Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, University of Southern California Keck School of Medicine, 1441 Eastlake Ave., MC9176, Los Angeles, California 90089-9176

Received 12 July 2007/ Returned for modification 21 August 2007/ Accepted 9 October 2007

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 T1 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.

* Corresponding author. Mailing address: USC Norris Comprehensive Cancer Ctr., Rm. 5428, 1441 Eastlake Ave., MC9176, Los Angeles, CA 90089-9176. Phone: (323) 865-0568. Fax: (323) 865-3019. E-mail: lieber{at}usc.edu

{triangledown} Published ahead of print on 22 October 2007.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} Present address: Microbiology and Molecular Genetics, Michigan State University, 5175 Biomedical Physical Sciences, East Lansing, MI 48824.

Molecular and Cellular Biology, January 2008, p. 50-60, Vol. 28, No. 1
0270-7306/08/$08.00+0     doi:10.1128/MCB.01251-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Wang, L., Wuerffel, R., Feldman, S., Khamlichi, A. A., Kenter, A. L. (2009). S region sequence, RNA polymerase II, and histone modifications create chromatin accessibility during class switch recombination. JEM 206: 1817-1830 [Abstract] [Full Text]  
  • Roy, D., Lieber, M. R. (2009). G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter. Mol. Cell. Biol. 29: 3124-3133 [Abstract] [Full Text]  
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