Biomedical subjects

Hideki Aihara

Publications and source records attributed to Hideki Aihara.

7 recordsLinked to original sources

PubMed2005

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination.

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.

Bacteriophage lambda↗

PubMed2005

A structural basis for allosteric control of DNA recombination by lambda integrase.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage lambda to integrate or excise its genome into and out of the host chromosome. lambda recombination is carried out by the bacteriophage-encoded integrase protein (lambda-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of lambda-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of lambda-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.

Allosteric Regulation↗

PubMed2005

Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution.

The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange. This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways. We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects. The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution.

Alleles↗

PubMed2004

Two structural features of lambda integrase that are critical for DNA cleavage by multimers but not by monomers.

Despite many years of genetic and biochemical studies on the lambda integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.

Amino Acid Substitution↗

PubMed2003

An NMR study on the interaction of Escherichia coli DinI with RecA-ssDNA complexes.

The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA. Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA. On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA- ATPgammaS, but had a slight effect on the structure of ssDNA-RecA-ATPgammaS complexes involving 8mer and 12mer ssDNA. We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA-RecA-ATPgammaS complex, together function to inhibit the co-protease activity of RecA.

Adenosine Triphosphate↗

PubMed2003

A conformational switch controls the DNA cleavage activity of lambda integrase.

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.

Bacteriophage lambda↗

PubMed2002

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase.

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.

Amino Acid Sequence↗