A continuing 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 and provided strong evidence that recombination of DNA strands was mediated by translocating Hin subunits between dimers. Future work will entail an ensemble of genetic, biochemical, and structural approaches that are directed at obtaining higher resolution views of the structural changes that occur upon synapsis and the mechanism(s) by which Fis-activates DNA catalysis and exchange. The crucial yet varied roles of accessory chromatin proteins has emerged as a second prominent theme of this work. Fis (Factor for Inversion Stimulation) was discovered because of its essential regulatory role in the Hin and Gin DNA inversion reactions, but it is now known to function in a large number of DNA reactions. We will investigate how Fis collaborates with the Xis protein of phage lambda to stimulate phage excision, with the ultimate goal of obtaining a detailed understanding of binding cooperativity and DNA structural changes that control the directionality of lambda site-specific recombination. Cellular Fis levels vary enormously with respect to growth phase and growth rates: Fis is the most abundant transcriptional regulator in E. coli under rapid growth conditions, whereas it is virtually absent in stationary phase. We will profile genome-wide effects of Fis on gene expression and protein levels under different growth conditions and molecularly dissect potentially novel regulatory units. Fis activates transcription by specifically interacting with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). X-ray crystal structures of Fis-DNA and Fis-alphaCTD-DNA complexes will be pursued. In addition to forming stable complexes at specific DNA binding sites, Fis also interacts nonspecifically with DNA at physiological concentrations and may play important roles in modulating chromosome structure because of its DNA-bending and looping activities. Fis effects on chromosome structure will be collaboratively addressed by single DNA molecule approaches as well as by bulk-phase in vitro and in vivo experiments.

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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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Portnoy, Matthew
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University of California Los Angeles
Schools of Medicine
Los Angeles
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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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