Purification of bacteriophage lambda repressor

Gao, Ning; Shearwin, Keith E.; Mack, John; Finzi, Laura; Dunlap, David

doi:10.1016/j.pep.2013.06.013

Cited by 10 publications

(6 citation statements)

References 31 publications

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“…S6). Dynamic, CI-mediated looping has been observed in tethered particle motion experiments in which there is no tension on the tether and no torsional control (11,30,31). Magnetic tweezers can be used to control torsion, but they also gently stretch the DNA (Fig.…”

mentioning

confidence: 99%

DNA supercoiling: A regulatory signal for the λ repressor

Ding¹,

Manzo²,

Fulcrand

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Topoisomerases, polymerases, and the chirality introduced by the binding of histones or nucleoid-associated proteins affect DNA supercoiling in vivo. However, supercoiling is not just a by-product of DNA metabolism. Supercoiling is an indicator of cell health, it modifies the accessibility of chromatin, and coordinates the transcription of genes. This suggests that regulatory, proteinmediated loops in DNA may sense supercoiling of the genome in which they are embedded. The λ repressor (CI) maintains the quiescent (lysogenic) transcriptome of bacteriophage λ in infected Escherichia coli. CI-mediated looping prevents overexpression of the repressor protein to preserve sensitivity to conditions that trigger virulence (lysis). Experiments were performed to assess how well the CI-mediated DNA loop traps superhelicity and determine whether supercoiling enhances CI-mediated DNA looping. CI oligomers partitioned plasmids into topological domains and prevented the passage of supercoiling between them. Furthermore, in single DNA molecules stretched and twisted with magnetic tweezers, levels of superhelical density confined in CI-mediated DNA loops ranged from −15% or +11%. Finally, in DNA under tensions that may occur in vivo, supercoiling lowered the free energy of loop formation and was essential for DNA looping. Supercoiling-enhanced looping can influence the maintenance of lysogeny in the λ repressor system; it can encode sensitivity to the energy level of the cell and creates independent topological domains of distinct superhelical density.DNA looping | DNA supercoiling | bacteriophage λ repressor | magnetic tweezer | transcription

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mentioning

confidence: 99%

DNA supercoiling: A regulatory signal for the λ repressor

Ding¹,

Manzo²,

Fulcrand

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Upon infection the phage can enter into either a silent life cycle or a virulent life cycle ( 29 ). This decision is, in part, controlled by a transcriptional repressor protein named CI ( 30 , 31 ). CI binds in two different promoter regions of the phage genome P R and P L ( 31 ).…”

Section: Resultsmentioning

confidence: 99%

“…This decision is, in part, controlled by a transcriptional repressor protein named CI ( 30 , 31 ). CI binds in two different promoter regions of the phage genome P R and P L ( 31 ). Each of these promoters comprises three different operator sites where CI binds as a dimer ( 30 , 31 ).…”

Section: Resultsmentioning

confidence: 99%

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Alignment of major-groove hydrogen bond arrays uncovers shared information between different DNA sequences that bind the same protein

Sedhom

Kinser

Solomon

2022

NAR Genomics and Bioinformatics

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Protein–DNA binding is of a great interest due to its importance in many biological processes. Previous studies have presented many factors responsible for the recognition and specificity, but understanding the minimal informational requirements for proteins that bind to multiple DNA-sites is still an understudied area of bioinformatics. Here we focus on the hydrogen bonds displayed by the target DNA in the major groove that take part in protein-binding. We show that analyses focused on the base pair identity may overlook key hydrogen bonds. We have developed an algorithm that converts a nucleotide sequence into an array of hydrogen bond donors and acceptors and methyl groups. It then aligns these non-covalent interaction arrays to identify what information is being maintained among multiple DNA sequences. For three different DNA-binding proteins, Lactose repressor, controller protein and λ-CI repressor, we uncovered the minimal pattern of hydrogen bonds that are common amongst all the binding sequences. Notably in the three proteins, key interacting hydrogen bonds are maintained despite nucleobase mutations in the corresponding binding sites. We believe this work will be useful for developing new DNA binding proteins and shed new light on evolutionary relationships.

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“…The wild type (WT) and mutant (D197G) lambda repressor (CI) proteins were expressed and purified as previously reported [ 13 ], and likely have activities of at least 50% [ 14 , 15 ]. Protein concentrations (both WT and D197G mutants) were varied between 0 and 500 nM for FCS or 200 and 800 nM for SFM experiments.…”

Section: Methodsmentioning

confidence: 99%

Specifically bound lambda repressor dimers promote adjacent non-specific binding

et al. 2018

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Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping.

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Purification of bacteriophage lambda repressor

Cited by 10 publications

References 31 publications

DNA supercoiling: A regulatory signal for the λ repressor

DNA supercoiling: A regulatory signal for the λ repressor

Alignment of major-groove hydrogen bond arrays uncovers shared information between different DNA sequences that bind the same protein

Specifically bound lambda repressor dimers promote adjacent non-specific binding

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