<i>Escherichia coli</i> integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA <i>in vitro</i>

Alazard, Robert; Bétermier, Mireille; Chandler, Michaël

doi:10.1111/j.1365-2958.1992.tb00895.x

Cited by 34 publications

(34 citation statements)

References 34 publications

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“…11,+6) vir3061 (-23,+13) proposed that binding of IHF to its cognate site located between O1 and 02 provides the right conformation for optimal repressor binding (Vogel et aI., 1991). This was confirmed by both in vivo and in vitro experiments which showed that IHF increases repression by repressor in vivo and facilitates repressor binding to the operators in vitro (Gama, Toussaint & Higgins, 1992;Alazard, B6termier & Chandler, 1992). The host H-NS protein, another nucleoid associated histone-like protein, has similar effects on repression and repressot binding as IHE Its exact mechanism of action is unknown and although it has been proposed that H-NS acts through protein-protein interactions with repressor (Falconi et al, 1991), the possibility remains open that H-NS binds DNA in the operator region, as it does for instance in the rrn promoters (Tippner et al, 1994).…”

Section: Lysogenysupporting

confidence: 75%

Regulation of bacteriophage Mu transposition

et al. 1994

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Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.

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Section: Lysogenysupporting

confidence: 75%

Regulation of bacteriophage Mu transposition

et al. 1994

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“…This 30% decrease may be the result of a fraction of StrRAG1 that binds non-specifically to DNA as well as the presence of a high concentration of polyanionic charge contributed by the phosphate groups of DNA, which may affect the overall stability of the complex in the electrophoretic field within the gel matrix. Gel-sieving effects combined with prolonged exposure of protein-DNA complexes to an electrophoretic field (3 h) and electrophoretic buffer may affect the results obtained using the EMSA methodology (40,41). These perturbations may especially affect the interactions between two macromolecular species when opposite charge attraction is a major factor in the stabilization of the complex.…”

Section: Resultsmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K D ‫؍‬ 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.The genes encoding the variable domains of immunoglobulins or T cell receptors are generated during lymphocyte differentiation by a somatic recombination reaction known as V(D)J recombination (1). The reaction is initiated by DNA double strand breaks created at the junction between two coding segments (termed V, D, or J) and their flanking recombination signal sequences (RSSs). 1 Cleavage is followed by a complex repair process that results in imprecise joining of the two coding segments and typically precise joining of the two RSSs. The RSSs consist of two conserved sequence elements, the heptamer (consensus 5Ј-CACAGTG-3Ј) and the nonamer (consensus 5Ј-ACAAAAACC-3Ј), separated by a poorly conserved spacer sequence of either 12 or 23 base pairs. Efficient recombination occurs only between a 12-RSS and a 23-RSS, a phenomenon known as the 12/23 rule (for review, see Ref.2). Recognition of 12/23-RSSs and concerted cleavage at the RSScoding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3 and 4), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5, 6). Binding and cleavage of DNA by the RAG1⅐RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins 8).Deletion mutagenesis has established the minimal "core" domains of murine RAG1 (residues 384 -1008 of the 1040 aa RAG1 protein; Fig. 1A) and RAG2 (residues 1-383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9 -12). Most bi...

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“…IHF plays an architectural role in the formation of various nucleoprotein complexes involved in different phenomena such as transcription activation, transcription repression, site-specific recombination, transposition, initiation of DNA replication, and plasmid partition. Binding of IHF could create a loop which stabilizes interaction of Roi with its binding site(s): this is reminiscent of the stabilization of the Mu repressor interaction with its operator (1,12). Interestingly, we noted the presence of an IHF binding site in the gene that encodes the related P1 Ant1 protein (from nt 855 to 867 in the sequence reported by Heisig et al [17]).…”

Section: Discussionmentioning

confidence: 76%

“…We speculated that IHF could bind the ihf site within the roi gene and possibly stabilize the interaction of the DNA-binding Roi protein with its target DNA, as happens in Mu repressor-operator interaction (1,12). The target of the Roi protein might then be located near the ihf site.…”

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confidence: 99%

Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants

Clerget

Boccard

1996

J Bacteriol

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Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth. The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q. This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif. We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site. By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene. We have isolated three independent phage mutants that are able to grow on an IHF ؊ host. They carry different mutations scattered in the roi gene and specifying single amino-acid changes. The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type. Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA. The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed.HK022 is a temperate coliphage, a member of the lambdoid family, to which belong phages lambda, 80, 21, P22, and 434. These phages share a common genetic organization which allows the exchange of information, genes or blocks of genes, among them (6). Some genes are always required for the phage to form plaques, while other genes are not essential for phage development in standard hosts but can play important roles in certain circumstances. These have been called accessory genes (6). Some accessory genes are carried by some of the phages in the family but not by others. It is known that phage lambda can form plaques in a host deficient in the integration host factor (IHF). In contrast, HK022 (8) and 80 (20, 21) do not form plaques in a host deficient in IHF. The basis of the HK022 IHF requirement was unknown and is the subject of this study.IHF is a small DNA-binding protein belonging to the family of prokaryotic histone-like proteins. It has remarkable DNAbinding properties: unlike most histone-like proteins, it binds preferentially to specific sites in DNA, makes most of its sequence-specific contacts in the minor groove of DNA, and is one of the strongest DNA-bending proteins (reviewed in references 22 and 23). In bacteria and their mobile genetic elements, IHF participates in a number of cellular processes such as DNA replication, site-specific recombination, bacteriophage packaging, and the positive and negative control of the expression of certain genes (reviewed in references 9, 10, and 23).Bacteriophages Mu, lambda cin-1, and 80 require IHF for plaque formation. The requirement of IHF for growth of Mu and lambda cin-1 appears to be satisfied by IHF's binding to a single identified site in each phage (14, 15). In both cases, IHF exerts its effect at the transcriptional level by modulating transcription from a promoter. For phage 80, a function (rha) which is inhibitory for growth of this phage in IHF Ϫ cells has been described; it maps in the QSR gene region of 80...

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Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro

Cited by 34 publications

References 34 publications

Regulation of bacteriophage Mu transposition

Regulation of bacteriophage Mu transposition

RAG1-DNA Binding in V(D)J Recombination

Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants

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