[Frontiers in Bioscience 8, d210-221, January 1, 2003]

THE PROBLEM OF HYPERNEGATIVE SUPERCOILING AND R-LOOP FORMATION IN TRANSCRIPTION

Marc Drolet, Sonia Broccoli, Fabien Rallu, Chadi Hraiky, Charles Fortin, Éric Massé and Imad Baaklini

Département de microbiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P.Québec, Canada, H3C 3J7

TABLE OF CONTENTS

1. Abstract
2. Introduction 3. DNA topoisomerase I and the regulation of global and local DNA supercoiling in Escherichia coli
3.1. Global supercoiling
3.2. Local supercoiling
3.3. Global and transcription-induced supercoiling in growth inhibition of topA null mutants
4. R-loop formation and hypernegative supercoiling
4.1. Biochemical and genetic evidence for R-loop formation in the absence of DNA topoisomerase I
4.2. Mechanisms of R-loop formation
4.2.1. R-loop initiation from the transcription bubble
4.2.2. R-loop initiation behind the moving RNA polymerase
4.3. Effects of translation inhibitors on hypernegative supercoiling and R-loop formation
5. Hypersupercoiling and R-loops in gene expression
5.1. R-loop formation and rRNA synthesis
5.2. R-loop formation and mRNA synthesis
6. Perspectives
7. Acknowledgments
8. References

1. ABSTRACT

DNA supercoiling and topoisomerases have long been known to affect transcription initiation. In many studies, topA mutants were used to perturb chromosomal supercoiling. Although such studies clearly revealed that supercoiling could significantly affect gene expression, they did not tell much about the essential function(s) of DNA topoisomerase I, encoded by topA. Indeed, the topA mutants used in these studies were growing relatively well, although this gene is normally essential for growth. These mutants were either carrying a topA allele with enough residual activity to permit growth, or if deleted for the topA gene, they were carrying a compensatory mutation allowing them to grow. We have recently used a set of isogenic strains carrying a conditional gyrB mutation that allowed us to study the real effects of losing topoisomerase I activity on cell physiology. The results of our work show that an essential function of topoisomerase I is related to transcription, more precisely to inhibit R-loop formation. This is in agreement with a series of biochemical studies that revealed a role for topoisomerase I in inhibiting R-loop formation during transcription in the presence of DNA gyrase. In addition, our studies may have revealed an important role for DNA supercoiling in modulating gene expression, not only at the level of transcription initiation but also during elongation. In this paper, we will first discuss global and local supercoiling, then we will address the topic of R-loop formation and finally, we will review the subject of hypersupercoiling and R-loop formation in gene expression. Whenever possible, we will try to make correlations with growth phenotypes, since such correlations reveal the essential function of DNA topoisomerase I.