The long-term goal of this project is to understand the mechanism that ensures lysogeny maintenance in temperate phages yet guaranteeing efficient switch to lysis when necessary. Such understanding will be useful in order to achieve a better control of phage-induced bacterial pathogenesis. It will also be valuable for manipulation of the inducibility set-point and use of phages in gene delivery applications. We will use ? bacteriophage as a model system. Recent findings showed that both stable lysogeny and efficient switch to lysis in ? rely on DNA loop formation by the lambda repressor protein CI. CI-mediated looping represents one of the simplest transcriptional regulatory feedback mechanisms and determines the choice of developmental growth by the phage. However, a characterization of CI-mediated looping is missing. The outcome of this research will also be pivotal for both our understanding of transcriptional regulation and multi-protein-mediated regulatoryloops. We have started investigating the molecular mechanism of ? looping and our results show: a pivotal role of the o3 sites for the thermodynamics of loop formation, a complex kinetics for both loop formation and breakdown, an important, CI concentration-dependent role of the o3 sites in aiding loop formation up to 20 nM CI, an important, CI concentration-independent role of the o3 sites in preventing loop rupture and, finally, CI non-specific binding. Together, these observations allow the formulation of a new hypothesis about the molecular mechanism for the formation and breakdown of the ? regulatory loop. This hypothesis suggests: (i) a "seeding" role for the CI dimers bound at the o3 sites in the "recruitment" of more dimers which may facilitate loop formation and interfere with loop breakdown;(ii) a physiological role for non-specifically bound CI dimers and their interaction. To test this hypothesis, we propose: (1) To understand the mechanism of CI-mediated loop formation and to identify the unlooped species relevant to it. We will do this by: (i) characterizing the different, relevant unlooped species and their dependence on CI concentration (Atomic Force Microscopy (AFM) and Tethered Particle Microscopy(TPM));(ii) quantifying the extent of CI non- specific binding and probing the possibility of cooperativity between non-specifically bound CI dimers (DNA pulling measurements by magnetic tweezers and theoretical modeling). (2) To elucidate the mechanism of CI-mediated loop breakdown, and characterization of the looped species relevant to it. We will do this by: (i) visualization of looped species and characterization of the dependence of their stoichiometry on time (AFM);(ii) characterization of the mechanism responsible for the time dependency of the kinetics of loop breakdown (AFM and TPM). (3) To investigate the effect of DNA supercoiling on CI-mediated looping (magnetic tweezers).

Public Health Relevance

The proposal aims to characterize the molecular bases of the epigenetic switch in lambda bacteriophage;an acute understanding of this mechanism will provide a better control of phage-induced bacterial pathogenesis and allow the use of inducible viruses for gene delivery and/or therapy. The lambda switch is also a paradigm of long-range interactions and multi-protein assemblages which if altered can lead to anomalies and tumors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084070-04
Application #
8269952
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2009-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
4
Fiscal Year
2012
Total Cost
$258,256
Indirect Cost
$91,639
Name
Emory University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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
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
Fernández-Sierra, Mónica; Shao, Qing; Fountain, Chandler et al. (2015) E. coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA. J Mol Biol 427:2305-18
Biton, Yoav Y; Kumar, Sandip; Dunlap, David et al. (2014) Lac repressor mediated DNA looping: Monte Carlo simulation of constrained DNA molecules complemented with current experimental results. PLoS One 9:e92475
Priest, David G; Kumar, Sandip; Yan, Yan et al. (2014) Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells. Proc Natl Acad Sci U S A 111:E4449-57
Priest, David G; Cui, Lun; Kumar, Sandip et al. (2014) Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and ýý repressors. Proc Natl Acad Sci U S A 111:349-54
Kumar, Sandip; Manzo, Carlo; Zurla, Chiara et al. (2014) Enhanced tethered-particle motion analysis reveals viscous effects. Biophys J 106:399-409
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
Gao, Ning; Shearwin, Keith; Mack, John et al. (2013) Purification of bacteriophage lambda repressor. Protein Expr Purif 91:30-6
Wang, Haowei; Dodd, Ian B; Dunlap, David D et al. (2013) Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor. Nucleic Acids Res 41:5746-56

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