Bacteriohpage lambda integrase is a site-specific recombinase that is responsible for viral integration during formation of lambda prophage. Studies on the Int system have been very informative as far as understanding the basis of lambda integration, but in a larger sense, they have also proven to be extremely insightful for the field of general homologous recombination in terms of understanding the molecular mechanism of formation and resolution of the Holliday junction (HJ). This four-armed crossed strand structure is considered to be the central intermediate formed during recombination. A great deal of what is known about the processing of the Holliday junction comes from studies on Int protein. It has been established that Int protein is a type I topoisomerase that carries out the cleavages and ligations responsible for recombination. Molecular details of DNA structure down to the nucleotide are known for Int protein interaction with the cognate binding site. Int acts on a core DNA target that has two binding sites for the protein arranged as an inverted repeat. Cleavage points on top and bottom strands are staggered and define the boundaries of an internal spacer region that is homologous in the recombining DNA partners. Recombination proceeds via a sequential pair of reciprocal strand exchanges that generate then resolve a 4-armed Holliday junction. Experimental support has accumulated for a trans cleavage model in which two Int protomers contribute different amino acid residues to generate a single active site. In other words, one protomer provides the domain for nucleotide positioning while the partner protomer contributes an active tyrosine as the nucleophile in the cleavage reaction. Experiments from Landy's laboratory have provided evidence more easily interpreted in terms of a single active site. Engineered suicide substrates in conjunction with an Int protein from a related lambda phage which has slightly different core DNA recognition have enabled experimentation showing that the same Int protomer bound makes the transient protein-DNA linkage.
The first aim analyzes protein-DNA interactions needed for resolution of HJs. While model wildtype HJs have four core Int binding sites and carry out the complete reaction, HJs with one and two sites are cleaved, but not resolved. Adding a third site permits resolution; the third molecule of Int is called the cross-core protomer. He will carry out several experiments to learn whether the active resolution complex has three or four Int protomers.
The second aim deals with identifying protein-protein interactions that influence the resolution reaction. One objective of these experiments is to try to resolve the apparent discrepancy between the finding of the Jayaram laboratory that Flp recombinanse cleaves in trans and the Landy laboratory result that Int cleaves in cis.
The third aim i s to define the minimal protein requirements for stimulation of resolution by a cross-core Int. Using the bispecific HJ with homologous partner sites, both lambda and HK022 Int proteins are required for efficient resolution; if lambda Y342F mutant Int is used, all of the resolution is at HK sites. That result will be extended by using fragments of lambda Int to stimulate HK022 Int at its cognate sites. The investigator notes that the most exciting outcome of these experiments would be to identify and characterize a peptide that allosterically activates Int cleavage. As an alternative these experiments could identify a peptide containing a trans nucleophile.
The fourth aim i s to learn how the DNA is configured in the HJ-protein complex. The configuration of the DNA arms in immobile 4-way HJs has been studied extensively in the absence of proteins. One set of experiments will determine the disposition of long and short arms in HJ- protein complexes by assaying gel mobility. Dr. Landy noted that preliminary studies indicate that existing methods for protein-free HJs can also be applied as proposed here. He will control the number and positions of bound Int by inserting non-binding sequences in various arms and by using bispecific Holliday junctions. He will also ask whether the disposition of the arms is altered after Int cleavage. The fifth aim is to learn which dynamic features are important for the formation and resolution of Holliday junctions. To do this, he will carry out careful kinetic measurements of resolution using different HJ substrates, in particular to learn whether the reaction is biphasic under certain conditions and how the ability to manipulate this fits with different views of the reaction. The sixth aim probes long-range effects of the att arm sites and accessory proteins on the efficiency and directionality of resolution. All of the previous aims deal with HJs that contain only the core Int sites; however, flanking the core are many other sequences that bind other molecules of Int plus molecules of other proteins such as IHF, Xis, and Fis. Thus, there are higher order structures in att site DNA that are important for recombination. Previous studies have shown that the basic architectural motif in these complexes is a bridge formed by the bivalent Int between a high-affinity arm site and a low-affinity core site. Formation of these bridges is facilitated by the DNA binding proteins which bring the correct pair of arm and core sites into close proximity and proper register.
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