A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site‐specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C‐terminal halves. A 40‐residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site‐specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well‐conserved C‐terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine‐433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.

The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction. The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region. Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region. We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites. There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites. When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.

A class of transcriptional regulator proteins bind to DNA at dyad-symmetric sites through a motif consisting of (i) a "leucine zipper" sequence that associates into noncovalent, parallel, alpha-helical dimers and (ii) a covalently connected basic region necessary for binding DNA. The basic regions are predicted to be disordered in the absence of DNA and to form alpha helices when bound to DNA. These helices bind in the major groove forming multiple hydrogen-bonded and van der Waals contacts with the nucleotide bases. To test this model, two peptides were designed that were identical to natural leucine zipper proteins only at positions hypothesized to be critical for dimerization and DNA recognition. The peptides form dimers that bind specifically to DNA with their basic regions in alpha-helical conformations.

Site-specific recombination between molecules of bacteriophage P1 DNA occurs at sites called loxP and requires the action of a protein that is the product of the P1 cre gene. Although recombination between two loxP sites is very efficient, recombination between loxP and a unique site in the bacterial chromosome (loxB) is inefficient and generates two hybrid lox sites called loxR and lozL. We present here the nucleotide sequences of all four lox sites. Analysis of these sequences indicates that (i) a region of extensive homology is not present at the loxP x loxB crossover point, in contrast to the 15-base pair common-core sequence in the bacteriophage A aft sites, and (ii) the sites contain a region ofdyad symmetry with 8-to 13-base pair inverted repeats separated by an 8-to 9-base pair sequence. The loxP X loB crossover point falls in the sequence that separates the inverted repeats, and deletions that remove either the left or the right inverted repeat of loxP inactivate the site. These two observations are consistent with the conclusion that the region of dyad symmetry is important in lox recombination. We have shown further that the loxP X loxP crossover point occurs in a 63-base pair sequence containing the loxP x loxB crossover point, suggesting that, despite the great difference in efficiencies of the two reactions, the crossover points may occur at the same place in both. Explanations for the different recombination properties of the various lox sites are discussed.Bacteriophage P1 is a temperate virus that has both lytic and lysogenic phases in its life cycle. In contrast to other temperate phages, such as A, P2, and P22, phage P1 rarely integrates into the chromosome of its host in its lysogenic mode but maintains itself as an autonomous unit-copy plasmid (1). Recently, the existence ofa site-specific recombination system in P1 has been demonstrated (2), and a number offunctions have been ascribed to it. Among these are the cyclization ofphage P1 DNA injected into a recA host and the rare integration ofP1 into the bacterial chromosome. Perhaps the most important role for this recombination, however, is to ensure proper segregation of P1 plasmid molecules to daughter cells at cell division (3). We have postulated that dimer plasmid molecules formed by homologous recombination between monomers interfere with orderly segregation ofthe products ofplasmid replication to daughter cells. P1 has overcome this problem by being able to resolve dimeric molecules rapidly into monomers by means of its site-specific recombination system.The site-specific recombination system of P1 consists of two elements, a site on the DNA called lox (for locus of crossingover) and a phage-encoded protein, the product of the P1 cre gene (4). The site on P1 DNA is termed loxP, and the site on the bacterial chromosome into which the P1 plasmid integrates with low efficiency is called loxB. On integration of P1 DNA The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be...

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Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg-Gly-Asp or Lys-Gly-Asp were discovered and synthesized. The cyclic, disulfide-bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds.

This study was designed to search for new regions of similarity in the integrase family of recombination proteins which consists of 28 members found in bacteria and yeast. A computer method based on an information content analysis has been used to align local regions of homology in the set of unaligned protein sequences from this family. Among the aligned regions with high information content were those containing the known conserved histidine, arginine and tyrosine residues. In addition, a new region was identified containing another arginine residue that appears to be conserved in all members of the family. To test further the importance of this newly identified arginine residue, mutants in the Cre protein from phase P1, a member of this integrase family, have been constructed which alter this residue. The mutations which change arginine to lysine and arginine to cysteine depress catalytic activity but not site-specific binding to the lox site. This result is expected for a conserved active site residue. This computer analysis also provides a means for searching for new members of the integrase family.

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