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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Janes, Daniel E
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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Chen, Wenyang; Mandali, Sridhar; Hancock, Stephen P et al. (2018) Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition. Elife 7:
Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R et al. (2017) Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase. J Bacteriol 199:
Kamar, Ramsey I; Banigan, Edward J; Erbas, Aykut et al. (2017) Facilitated dissociation of transcription factors from single DNA binding sites. Proc Natl Acad Sci U S A 114:E3251-E3257
Hadizadeh, Nastaran; Johnson, Reid C; Marko, John F (2016) Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome. J Bacteriol 198:1735-42
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
Johnson, Reid C (2015) Site-specific DNA Inversion by Serine Recombinases. Microbiol Spectr 3:MDNA3-0047-2014
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
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
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

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