MCB Accepts, published online ahead of print on 19 October 2009

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Mol. Cell. Biol. doi:10.1128/MCB.00897-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Competition Between the RNA Transcript and the Nontemplate DNA Strand During R-Loop Formation In Vitro: A Nick Can Serve as a Strong R-loop Initiation Site

Deepankar Roy, Zheng Zhang, Zhengfei Lu, Chih-Lin Hsieh, and Michael R. Lieber^*

Departments of Pathology, of Biochemistry & Molecular Biology, of Molecular Microbiology & Immunology, of Urology, and of Biological Sciences (Section of Molecular & Computational Biology), USC Norris Comprehensive Cancer Ctr., Rm. 5428, 1441 Eastlake Ave., MC9176, Los Angeles, CA 90089-9176

^* To whom correspondence should be addressed. Email: lieber{at}usc.edu.

Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template DNA strand, resulting in an R-loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone (RIZ) is favored by G-clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance between the promoter and the R-loop supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA transcript is downstream from the 5' end of the transcript, the ability of this portion of the transcript to anneal to the template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases because the G-rich portion of the RNA is now closer to the 5' end of the transcript. Short G-clustered regions can act as RIZs and reduce the distance-induced suppression on R-loop formation. Supercoiled DNA is known to favor transient separation of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick can serve as a strong R-loop initiation zone (RIZ), even in regions with no G-richness. This has important implications for class switch recombination and somatic hypermutation, and possibly for other biological processes in transcribed regions.