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Molecular and Cellular Biology, June 2009, p. 3124-3133, Vol. 29, No. 11
0270-7306/09/$08.00+0     doi:10.1128/MCB.00139-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter{triangledown} ,{dagger}

Deepankar Roy and Michael R. Lieber*

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

Received 30 January 2009/ Returned for modification 27 February 2009/ Accepted 13 March 2009

R-loops form cotranscriptionally in vitro and in vivo at transcribed duplex DNA regions when the nascent RNA is G-rich, particularly with G clusters. This is the case for phage polymerases, as used here (T7 RNA polymerase), as well as RNA polymerases in bacteria, Saccharomyces cerevisiae, avians, mice, and humans. The nontemplate strand is left in a single-stranded configuration within the R-loop region. These structures are known to form at mammalian immunoglobulin class switch regions, thus exposing regions of single-stranded DNA for the action of AID, a single-strand-specific cytidine deaminase. R-loops form by thread-back of the RNA onto the template DNA strand, and here we report that G clusters are extremely important for the initiation phase of R-loop formation. Even very short regions with one GGGG sequence can initiate R-loops much more efficiently than random sequences. The high efficiencies observed with G clusters cannot be achieved by having a very high G density alone. Annealing of the transcript, which is otherwise disadvantaged relative to the nontemplate DNA strand because of unfavorable proximity while exiting the RNA polymerase, can offer greater stability if it occurs at the G clusters, thereby initiating an R-loop. R-loop elongation beyond the initiation zone occurs in a manner that is not as reliant on G clusters as it is on a high G density. These results lead to a model in which G clusters are important to nucleate the thread-back of RNA for R-loop initiation and, once initiated, the elongation of R-loops is primarily determined by the density of G on the nontemplate DNA strand. Without both a favorable R-loop initiation zone and elongation zone, R-loop formation is inefficient.

* 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 23 March 2009.

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

Molecular and Cellular Biology, June 2009, p. 3124-3133, Vol. 29, No. 11
0270-7306/09/$08.00+0     doi:10.1128/MCB.00139-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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