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J. Biol. Chem., Vol. 283, Issue 19, 12756-12762, May 9, 2008

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G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II^*

Silvia Tornaletti¹, Shaun Park-Snyder, and Philip C. Hanawalt

From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020 and the Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ⁺ strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

Received for publication, June 18, 2007 , and in revised form, December 10, 2007.

^* This work was supported, in whole or in part, by National Institutes of Health Grant CA-77712 from NCI, United States Department of Health and Human Services (to S. T. and P. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610. Tel.: 352-392-3329; Fax: 352-392-3305; E-mail: silviat{at}ufl.edu.