For decades, it has been recognized that transcribing along the DNA double helix by a RNA polymerase can enhance localized DNA supercoiling. This process has been elegantly explained by a """"""""twin-supercoiled- domain"""""""" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase and negative supercoils behind it. However, detailed mechanisms at the molecular level are still unavailable. For instance, how DNA topological barriers affect transcription-coupled DNA supercoiling (TCDS) has not been fully understood. The long-term goal of the proposed research is to understand the molecular mechanisms and biological functions of TCDS and DNA topological barriers. The objectives of this application are to determine what properties make certain DNA-looping and -wrapping proteins function as topological barriers and block supercoil diffusion, to study how DNA topological barriers regulate TCDS in E. coli cells, and to examine how localized, transient TCDS activates gene expression in vivo. Our central hypothesis is that TCDS by RNA polymerases is a major chromosome remodeling force in E. coli cells where the cells use nucleoprotein-based, topological barriers to confine TCDS to localized regions and, as a result, greatly influence the nearby, coupled DNA transactions. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory and will be tested by pursuing the following three specific aims: 1) to determine molecular mechanisms of DNA topological barriers and examine how the DNA topological barriers confine supercoils to localized regions and affect the efficiency of TCDS;2) to examine the role of DNA topological barriers in TCDS in E. coli cells;3) to investigate how transient, dynamic TCDS activates transcription in E. coli cells and how DNA topological barriers affect the efficiency of transcription activation by TCDS. Information from the proposed experiments will ultimately provide us with a better understanding of the mechanisms of TCDS, especially the roles of DNA topological barriers in TCDS and the coupled gene transcription and expression.

Public Health Relevance

Almost all DNA molecules inside cells including human cells are supercoiled. In fact, DNA supercoiling affects all DNA transactions, i.e., DNA replication, recombination, and transcription, and greatly influences genomic stability and susceptibility to cancer and certain hereditary diseases, such as fragile X syndrome, Huntington's disease, and autism. The significance of this research stems from its potential to provide a basis for better understanding of essential biological processes: gene transcription and expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM109254-01A1
Application #
8762585
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Carter, Anthony D
Project Start
2014-09-01
Project End
2017-08-31
Budget Start
2014-09-01
Budget End
2017-08-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Florida International University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33199
Fulcrand, Geraldine; Dages, Samantha; Zhi, Xiaoduo et al. (2016) DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli. Sci Rep 6:19243
Ahmad, Muzammil; Xue, Yutong; Lee, Seung Kyu et al. (2016) RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals. Nucleic Acids Res 44:6335-49
Fulcrand, Geraldine; Chapagain, Prem; Dunlap, David et al. (2016) Direct observation of a 91 bp LacI-mediated, negatively supercoiled DNA loop by atomic force microscope. FEBS Lett 590:613-8
Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo et al. (2016) Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis. Phytochemistry 122:11-21
Gu, Maxwell; Berrido, Andrea; Gonzalez, Walter G et al. (2016) Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases. Sci Rep 6:36006
Frost, Lorraine; Baez, Maria A M; Harrilal, Christopher et al. (2015) The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2). PLoS One 10:e0130478
Alonso, Nicole; Guillen, Roboan; Chambers, Jeremy W et al. (2015) A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions. Nucleic Acids Res 43:e52
Ding, Yue; Manzo, Carlo; Fulcrand, Geraldine et al. (2014) DNA supercoiling: a regulatory signal for the λ repressor. Proc Natl Acad Sci U S A 111:15402-7