A long term goal of this project is to understand the assembly, control, and function of nucleoprotein complexes that promote Hin-catalyzed site-specific DNA inversion. Work during the past funding period established the overall structure of the tetrameric catalytic core of the Hin synaptic complex, provided strong evidence that recombination of DNA strands was mediated by rotation of Hin subunit pairs within the tetramer, and showed how the Fis/enhancer system together with DNA supercoiling controls subunit rotation. Future work will entail an ensemble of genetic, biochemical, and structural approaches and will emphasize the mechanism by which the Fis/enhancer element activates initial reaction steps as well as controls the subunit rotation process. Mechanistic analysis will commence on an enzyme from a different class of serine recombinase, emphasizing its novel properties. A prominent theme of the project concerns the DNA binding properties and regulatory roles of nucleoid proteins on recombination and transcription reactions. New aspect on the control of phage lambda site specific recombination by nucleoid proteins will be pursued. X- ray crystal structure analyses of complexes bound by the nucleoid protein Fis will be extended to higher- order complexes which contain the phage lambda Xis protein and the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). These structural studies will provide new information on the role of DNA conformational flexibility on indirect recognition and cooperative DNA binding by proteins. Cellular Fis levels vary enormously with respect to growth phase and growth rates, being the most abundant DNA binding protein in E. coli under rapid growth conditions but virtually absent in stationary phase. Mechanisms of transcriptional regulation by Fis through specific and non-specific DNA binding will be investigated. Recent in vitro evidence suggests Fis may play an important role in modulating chromosome structure because of its DNA-bending and looping activities. Fis effects on chromosome structure will be addressed by a combination of in vivo and in vitro approaches, including collaborative single-DNA molecule approaches as well as by bulk-phase in vitro and in vivo experiments.

Public Health Relevance

This research aims to understand the mechanism and control of specialized DNA recombination reactions, particulariy those of the less well understood serine recombinase family. A broader goal is to understand how abundant non-histone DNA binding and bending proteins modulate chromosome structure and control DNA recombination and gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM038509-28
Application #
8692808
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Janes, Daniel E
Project Start
1987-07-01
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
28
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Hancock, Stephen P; Stella, Stefano; Cascio, Duilio et al. (2016) DNA Sequence Determinants Controlling Affinity, Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis. PLoS One 11:e0150189
Hadizadeh, Nastaran; Johnson, Reid C; Marko, John F (2016) Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome. J Bacteriol 198:1735-42
Johnson, Reid C (2015) Site-specific DNA Inversion by Serine Recombinases. Microbiol Spectr 3:MDNA3-0047-2014
Giuntoli, Rebecca D; Linzer, Nora B; Banigan, Edward J et al. (2015) DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response. J Mol Biol 427:3123-36
Chang, Yong; Johnson, Reid C (2015) Controlling tetramer formation, subunit rotation and DNA ligation during Hin-catalyzed DNA inversion. Nucleic Acids Res 43:6459-72
Mandali, Sridhar; Dhar, Gautam; Avliyakulov, Nuraly K et al. (2013) The site-specific integration reaction of Listeria phage A118 integrase, a serine recombinase. Mob DNA 4:2
McLean, Meghan M; Chang, Yong; Dhar, Gautam et al. (2013) Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion. Elife 2:e01211
Hancock, Stephen P; Ghane, Tahereh; Cascio, Duilio et al. (2013) Control of DNA minor groove width and Fis protein binding by the purine 2-amino group. Nucleic Acids Res 41:6750-60
Hadizadeh Yazdi, Nastaran; Guet, Calin C; Johnson, Reid C et al. (2012) Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions. Mol Microbiol 86:1318-33
Nafissi, Maryam; Chau, Jeannette; Xu, Jimin et al. (2012) Robust translation of the nucleoid protein Fis requires a remote upstream AU element and is enhanced by RNA secondary structure. J Bacteriol 194:2458-69

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