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1.
EMBO J ; 20(5): 1203-12, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11230143

ABSTRACT

lambda Integrase (Int) has the distinctive ability to bridge two different and well separated DNA sequences. This heterobivalent DNA binding is facilitated by accessory DNA bending proteins that bring flanking Int sites into proximity. The regulation of lambda recombination has long been perceived as a structural phenomenon based upon the accessory protein-dependent Int bridges between high-affinity arm-type (bound by the small N-terminal domain) and low-affinity core-type DNA sites (bound by the large C-terminal domain). We show here that the N-terminal domain is not merely a guide for the proper positioning of Int protomers, but is also a context-sensitive modulator of recombinase functions. In full-length Int, it inhibits C-terminal domain binding and cleavage at the core sites. Surprisingly, its presence as a separate molecule stimulates the C-terminal domain functions. The inhibition in full-length Int is reversed or overcome in the presence of arm-type oligonucleotides, which form specific complexes with Int and core-type DNA. We consider how these results might influence models and experiments pertaining to the large family of heterobivalent recombinases.


Subject(s)
Bacteriophage lambda/enzymology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Integrases/chemistry , Integrases/metabolism , Recombination, Genetic , Binding Sites , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Electrophoresis, Agar Gel , Nucleic Acid Conformation , Oligodeoxyribonucleotides , Peptide Fragments , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins , Substrate Specificity , Topoisomerase I Inhibitors
3.
J Mol Biol ; 279(3): 513-27, 1998 Jun 12.
Article in English | MEDLINE | ID: mdl-9641975

ABSTRACT

Escherichia coli phage lambda integrase (Int) is a 40 kilodalton, 356 amino acid residue protein, which belongs to the lambda Int family of site-specific recombinases. The amino-terminal domain (residues 1 to 64) of Int binds to "arm-type" DNA sites, distant from the sites of DNA cleavage. The carboxy-terminal fragment, termed C65 (residues 65 to 356), binds "core-type" DNA sites and catalyzes cleavage and ligation at these sites. It has been further divided into two smaller domains, encompassing residues 65 to 169 and 170 to 356, respectively. The latter has been characterized and its crystal structure has been determined. Although this domain catalyzes the cleavage and rejoining of DNA strands it, unexpectedly, does not form electrophorectically stable complexes with core-type DNA. Here we have investigated the critical features of lambda Int binding to core-type DNA sites; especially, the role of the central 65 to 169 domain. To eliminate the complexities arising from lambda Int's heterobivalency we studied Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core-type sites. Zero-length UV crosslinking was used to show that Ala125 and Ala126 make close contact with bases in the core-type DNA. Modification by pyridoxal 5'-phosphate was used to identify Lys103 at the protein-DNA interface. Since both of the identified loci are in the central domain, it was cloned and purified and found to bind to core-type DNA autonomously and specifically. The synergistic roles of the catalytic and the central, or core-binding (CB), domains in the interaction with core-type DNA are discussed for (Int and related DNA recombinases.


Subject(s)
Bacteriophage lambda/enzymology , Coliphages/enzymology , DNA/metabolism , Integrases/chemistry , Binding Sites/genetics , Cross-Linking Reagents/metabolism , DNA-Binding Proteins/chemistry , Kinetics , Lysine/metabolism , Metalloendopeptidases/metabolism , Oligodeoxyribonucleotides/metabolism , Peptide Fragments/chemistry , Pyridoxal Phosphate/metabolism , Ultraviolet Rays
4.
Nucleic Acids Res ; 26(2): 391-406, 1998 Jan 15.
Article in English | MEDLINE | ID: mdl-9421491

ABSTRACT

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).


Subject(s)
DNA Nucleotidyltransferases/chemistry , Integrases/chemistry , Amino Acid Sequence , Conserved Sequence , DNA Mutational Analysis , DNA Nucleotidyltransferases/metabolism , Humans , Infant, Newborn , Integrases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Recombination, Genetic , Sequence Alignment , Structure-Activity Relationship
5.
J Mol Biol ; 272(4): 493-508, 1997 Oct 03.
Article in English | MEDLINE | ID: mdl-9325107

ABSTRACT

lambda Site-specific recombination requires a short stretch of sequence homology that might be sensed during strand swapping, during ligation and/or during isomerization of the obligate Holliday junction intermediate. Here, we use half-att site suicide substrates to study single and double top-strand-transfers, isolated from the subsequent steps of the reaction. The double-strand-transfer is analogous to a top-strand exchange and consists of one normal top-strand and one "contrary" bottom-strand to top-strand ligation between the half-att site substrate and its full-site partner. The resulting covalent three-way DNA junctions are poor substrates for resolution in the forward or reverse direction. We show that both the rate and the efficiency of Y-junction formation are homology dependent. Pairing of three nucleotides (either in the forward or in the contrary alignment) provides maximal stability to strand swapping. Complementary base-pairing next to one top-strand site (with or without ligation) stimulates strand-transfer at the other mismatched site. The data suggest that homology can be sensed at the strand-swapping step before ligation. However, homology also stimulates ligation and stabilizes the products, as is evident from the different rates of closed Y-junction formation in the presence or absence of homology. Furthermore, under recombination conditions, single top-strand-transfers are subject to reversal even in the presence of sequence homology; stability depends on a double-strand-transfer, i.e. dissociation of covalent Int.


Subject(s)
Bacteriophage lambda/genetics , Recombination, Genetic , Bacterial Proteins/metabolism , DNA, Viral/metabolism , Integrases/metabolism , Membrane Proteins/metabolism , Sequence Homology, Nucleic Acid
6.
Proc Natl Acad Sci U S A ; 94(12): 6104-9, 1997 Jun 10.
Article in English | MEDLINE | ID: mdl-9177177

ABSTRACT

The Escherichia coli phage lambda integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that lambda Int consists of three functional and structural domains: residues 1-64 specify recognition of "arm-type" DNA sequences distant from the region of strand exchange; residues 65-169 contribute to specific recognition of "core-type" sequences at the sites of strand exchange and possibly to protein-protein interactions; and residues 170-356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.


Subject(s)
Bacteriophage lambda/enzymology , DNA Nucleotidyltransferases/chemistry , DNA Nucleotidyltransferases/metabolism , Integrases/chemistry , Integrases/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Crystallography, X-Ray , Endopeptidases , Escherichia coli/virology , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Point Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinases , Tyrosine
7.
EMBO J ; 16(12): 3744-55, 1997 Jun 16.
Article in English | MEDLINE | ID: mdl-9218815

ABSTRACT

Lambda site-specific recombination proceeds by a pair of sequential strand exchanges that first generate and then resolve a Holliday junction intermediate. A family of synthetic Holliday junctions with the branch point constrained to the center of the 7 bp overlap region was used to show that resolution of the top strands and resolution of the bottom strands are symmetrical but stereochemically distinct processes. Lambda integrase is sensitive to isomeric structure, preferentially resolving the pair of strands that are crossed in the protein-free Holliday junction. At the branch point of stacked immobile Holliday junctions, the number of purines is preferentially maximized in the crossed (versus continuous) strands if there is an inequality of purines between strands of opposite polarity. This stacking preference was used to anticipate the resolution bias of freely mobile junctions and thereby to reinforce the conclusions with monomobile junctions. The results provide a strong indication that in the complete recombination reaction a restacking of helices occurs between the top and bottom strand exchanges.


Subject(s)
Bacteriophage lambda/enzymology , DNA/metabolism , Integrases/metabolism , Nucleic Acid Conformation , DNA/chemistry , DNA, Recombinant/chemistry , DNA, Recombinant/metabolism , Isomerism , Purines
8.
Science ; 276(5309): 126-31, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9082984

ABSTRACT

Lambda integrase is archetypic of site-specific recombinases that catalyze intermolecular DNA rearrangements without energetic input. DNA cleavage, strand exchange, and religation steps are linked by a covalent phosphotyrosine intermediate in which Tyr342 is attached to the 3'-phosphate of the DNA cut site. The 1.9 angstrom crystal structure of the integrase catalytic domain reveals a protein fold that is conserved in organisms ranging from archaebacteria to yeast and that suggests a model for interaction with target DNA. The attacking Tyr342 nucleophile is located on a flexible loop about 20 angstroms from a basic groove that contains all the other catalytically essential residues. This bipartite active site can account for several apparently paradoxical features of integrase family recombinases, including the capacity for both cis and trans cleavage of DNA.


Subject(s)
Bacteriophage lambda/enzymology , DNA/metabolism , Integrases/chemistry , Protein Conformation , Recombination, Genetic , Amino Acid Sequence , Attachment Sites, Microbiological , Binding Sites , Cloning, Molecular , Conserved Sequence , Crystallography, X-Ray , DNA Nucleotidyltransferases/chemistry , DNA Nucleotidyltransferases/metabolism , Hydrogen Bonding , Integrases/metabolism , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Recombinases , Tyrosine/chemistry , Tyrosine/metabolism , Virus Integration
9.
Structure ; 5(2): 153-7, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9032076

ABSTRACT

The crystal structure of integration host factor (IHF) complexed with DNA shows how a small heterodimeric protein can induce a big bend in DNA. IHF exerts leverage in the minor groove and wraps DNA around the body of the protein, providing another example of sequence-specific recognition of the minor groove.


Subject(s)
Bacterial Proteins/metabolism , DNA, Bacterial/chemistry , DNA-Binding Proteins/metabolism , Nucleic Acid Conformation , Amino Acid Sequence , Bacterial Proteins/chemistry , DNA, Bacterial/metabolism , DNA-Binding Proteins/chemistry , Integration Host Factors , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship
10.
J Biol Chem ; 271(47): 29599-604, 1996 Nov 22.
Article in English | MEDLINE | ID: mdl-8939889

ABSTRACT

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket. The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.


Subject(s)
Bacteriophage lambda/enzymology , Ethylmaleimide/pharmacology , Integrase Inhibitors/pharmacology , Integrases/metabolism , Amino Acid Sequence , Cysteine/metabolism , Integrases/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed
11.
Mol Microbiol ; 21(1): 69-76, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8843435

ABSTRACT

Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.


Subject(s)
Bacteriophage lambda/enzymology , Bacteriophage lambda/genetics , Codon/genetics , Integrases/biosynthesis , Integrases/genetics , RNA, Transfer, Arg/genetics , Amino Acid Sequence , Base Sequence , DNA, Viral/genetics , Escherichia coli/genetics , Frameshift Mutation , Gene Expression Regulation, Viral , Genes, Bacterial , Genes, Viral , Protein Biosynthesis
12.
J Mol Biol ; 253(2): 228-42, 1995 Oct 20.
Article in English | MEDLINE | ID: mdl-7563085

ABSTRACT

The prokaryotic integration host factor (IHF) is a DNA-bending protein that binds to specific DNA sites as a heterodimer. Genetic and mutational analyses have previously identified asymmetric protein-DNA contacts by the individual subunits. By exploiting the unique sequence and positional context of one IHF binding site, H' in Lambda attachment sites (att sites), we have identified a symmetry element of binding and have localized the functional bend center to the center of this symmetry. A shift of the H' bend center by a single base-pair to the right or to the left within the very tight loop formed with Lambda integrase (Int) and IHF in att-site "intasomes" severely reduces recombination. This suggests that a precise, but wrongly positioned, DNA bend within a loop of constant length negatively influences the juxtaposition or "phasing" of the core-type and arm-type Int binding sites by differentially affecting the length of each leg of the loop. Furthermore, ten base-pair insertions within this loop that should not interfere with correct helical phasing are sensed in a position-dependent manner. Distal insertions abolish recombination, whereas proximal or double insertions (in both legs of the loop) are well tolerated.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Nucleic Acid Conformation , Plasmids/chemistry , Base Composition , Base Sequence , Binding Sites , DNA-Binding Proteins/chemistry , Integration Host Factors , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Insertional , Plasmids/metabolism , Protein Conformation , Recombination, Genetic , Restriction Mapping
13.
Curr Biol ; 5(2): 139-48, 1995 Feb 01.
Article in English | MEDLINE | ID: mdl-7743177

ABSTRACT

BACKGROUND: Many site-specific recombinases act by forming and resolving branched Holliday junction intermediates. Previous findings have been consistent with models involving branch migration across the 'overlap region' of obligate homology, located between the staggered sites where the two single-strand exchanges occur. We have investigated the validity of such models in the case of bacteriophage lambda site-specific recombination. RESULTS: By using synthetic lambda att-site Holliday junctions, incorporating sequence heterologies that impose constraints on branch migration, we have found that the optimal position of the junction for either top-strand or bottom-strand resolution by lambda integrase (Int) is not at the ends, but close to the middle of the seven base-pair overlap region. A minor shift of the branch point around the central base pair caused a remarkable switch in resolution bias. Our findings suggest that branch migration is limited to the central one to three base pairs of the overlap region. They lead to a new model for lambda site-specific recombination, in which there are two symmetrical swaps of two to three nucleotides each, linked by a central isomerization step that causes a change of the stacking interactions between the four junction arms. On the basis of isolated strand-joining reactions carried out by Int in the presence or absence of base complementarity, we propose that sequence homology is sensed during the annealing step prior to strand joining. The new model eliminates mechanistic complications associated with large helical rotations required by branch-migration models. CONCLUSIONS: The results reported here suggest that the recognition of sequence homology in Int-dependent site-specific recombination does not rely primarily on branch migration. The property of cleaving Holliday junctions a few base pairs away from the crossover puts lambda Int into the same category as endonucleases that cleave Holliday junctions in homologous recombination.


Subject(s)
Bacteriophage lambda/genetics , DNA, Viral/genetics , Recombination, Genetic , Sequence Homology, Nucleic Acid , Base Sequence , DNA Nucleotidyltransferases/metabolism , Integrases , Molecular Sequence Data
14.
EMBO J ; 14(2): 397-406, 1995 Jan 16.
Article in English | MEDLINE | ID: mdl-7835349

ABSTRACT

In lambda site-specific recombination, the integrative and excisive reactions proceed via two different Holliday junction intermediates, both of which are generated and resolved by a pair of sequentially ordered single strand exchanges. Factors affecting the directionality and efficiency of the second pair of strand exchanges were examined using artificial Holliday junctions (chi-forms). The integrative and excisive recombination intermediates respond differently to the accessory DNA bending proteins integration host factor and excisionase (Xis). These differences between the two recombination intermediates result from a different interaction pattern between proteins binding to the left (P arm) and right (P' arm) of the crossover region. The effect of Xis protein on the directionality of resolution, i.e. the choice of which strands are exchanged, is consistent with a role in promoting the second strand exchange during excision. Proteins binding to the left of the crossover region (P arm) primarily influence the directionality of resolution, while proteins binding to the right (P' arm) have a greater effect on the overall efficiency of resolution. Together, the effect of proteins binding to sites in the P and P' arms is to greatly enhance resolution of the two different Holliday intermediates and to favor resolution in the 'forward' direction for both integrative and excisive recombination.


Subject(s)
Bacteriophage lambda/genetics , DNA Nucleotidyltransferases/metabolism , DNA-Binding Proteins/metabolism , Recombination, Genetic , Viral Proteins , Bacteriophage lambda/metabolism , Nucleic Acid Conformation
15.
EMBO J ; 13(18): 4421-30, 1994 Sep 15.
Article in English | MEDLINE | ID: mdl-7925285

ABSTRACT

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.


Subject(s)
Bacteriophage lambda/enzymology , DNA Nucleotidyltransferases/metabolism , DNA/metabolism , DNA Replication , Integrases , Models, Genetic , Nucleic Acid Conformation , Protein Binding , Recombination, Genetic , Substrate Specificity , Virus Integration
16.
J Biol Chem ; 269(32): 20798-805, 1994 Aug 12.
Article in English | MEDLINE | ID: mdl-8051182

ABSTRACT

We have defined the bacterial and viral DNA targets (att sites) of P22 site-specific recombination and characterized their interaction with integrase (Int) protein. The bacterial DNA target, attB, is approximately 27 base pairs and consists of two core type Int binding sites as inverted repeats. The top and bottom Int cleavage sites fall within the core type Int binding sites and are separated by a 7-base pair overlap region. A similar core region is found in the viral DNA target, attP, which is approximately 260 base pairs long and contains two IHF binding sites and five arm type binding sites for Int. The results suggest that P22 Int, like lambda Int, is a heterobivalent DNA-binding protein that is capable of forming complex higher order structures with recombinogenic function. Although P22 and lambda recombination involve very similar multiprotein interactions and core region structures, there are significant differences in the arrangements of distal protein binding sites. These differences are discussed in terms of the possible flexibility of the Int protein and the specificity with which the higher order complexes assemble and/or function.


Subject(s)
Bacteriophage P22/chemistry , DNA Nucleotidyltransferases/chemistry , Attachment Sites, Microbiological , Bacteriophage P22/physiology , Base Sequence , Binding Sites , Conserved Sequence , DNA, Recombinant , Hydrolysis , Integrases , Molecular Sequence Data
17.
EMBO J ; 13(11): 2714-24, 1994 Jun 01.
Article in English | MEDLINE | ID: mdl-8013469

ABSTRACT

A reciprocal strand exchange between two DNA helices generates the crossed-strand intermediate, or Holliday junction, which is common to many pathways of homologous and site-specific recombination. The Int family of recombinases are unique in their ability to both make and resolve Holliday junctions. Previous experiments utilizing 'synthetic' att site Holliday junctions to study the mechanisms associated with the cleavage, transfer and ligation of DNA strands have been confined to studying reciprocal strand exchanges (a pair of temporally overlapping strand cleavages). To circumvent this limitation, we have designed synthetic suicide Holliday junctions that make it possible to monitor individual DNA strand cleavage events. These substrates contain a pre-existing nick in the vicinity of the Int binding site; when Int introduces a second nick into these substrates, the 5'OH nucleophile required for ligation (in either the forward or reverse reaction) is lost by diffusion, thus trapping the covalent protein-DNA intermediate. The results indicate that resolution (involving two partner Ints) is stimulated by additional 'cross-core' Ints as a result of enhanced cleavage rates, and not as a result of enhanced co-ordination of cleavage. Several models for the role of the 'cross-core' Ints during resolution are discussed, as well as the usefulness of these substrates for studying additional aspects of the Holliday junction resolution reaction.


Subject(s)
DNA Nucleotidyltransferases/metabolism , DNA, Viral/metabolism , Models, Genetic , Recombination, Genetic/genetics , Attachment Sites, Microbiological/genetics , Base Sequence , Binding Sites , DNA, Viral/chemistry , Integrases , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation
18.
Curr Opin Genet Dev ; 3(5): 699-707, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8274851

ABSTRACT

This review focuses on two of the approximately 30 members of the diverse Int family of site-specific recombinases. The lambda recombination system represents those reactions involving accessory proteins and a complex higher-order structure. The FLP system represents the most streamlined reactions and has been the subject of detailed and informative studies on the mechanisms of DNA cleavage and ligation.


Subject(s)
DNA Nucleotidyltransferases/genetics , Fungal Proteins/genetics , Recombination, Genetic , Viral Proteins/genetics , DNA Nucleotidyltransferases/chemistry , Fungal Proteins/chemistry , Integrases , Viral Proteins/chemistry
19.
Science ; 256(5054): 198-203, 1992 Apr 10.
Article in English | MEDLINE | ID: mdl-1533056

ABSTRACT

The excisive recombination reaction of bacteriophage lambda involves a specific and efficient juxtaposition of two distant higher order protein-DNA complexes on the chromosome of Escherichia coli. These complexes, which mediate synapsis and strand exchange, consist of two DNA sequences, attL and attR, the bivalent DNA binding protein Int, and the sequence-specific DNA bending proteins, IHF, Xis, and Fis. The protein-protein and protein-DNA interactions within, and between, these complexes were studied by various biochemical techniques and the patterns of synergism among pairs of mutants with marginally impaired recombination function were analyzed. The DNA bending proteins facilitated long-range tethering of high- and low-affinity DNA sites by the bivalent Int protein, and a specific map is proposed for the resulting Int bridges. These structural motifs provide a basis for postulating the mechanism of site-specific recombination and may also be relevant to other pathways in which two distant chromosomal sites become associated.


Subject(s)
Bacteriophage lambda/enzymology , Chromosomes, Bacterial , DNA Nucleotidyltransferases/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Base Sequence , Chromosome Mapping , Crosses, Genetic , DNA, Bacterial/genetics , Escherichia coli/metabolism , Genes, Bacterial , Genetic Linkage , Integrases , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Plasmids , Protein Conformation , Recombination, Genetic
20.
Proc Natl Acad Sci U S A ; 88(2): 588-92, 1991 Jan 15.
Article in English | MEDLINE | ID: mdl-1824874

ABSTRACT

The virally encoded Xis protein is one of the components in the site-specific recombination reactions of bacteriophage lambda. It is required for excisive recombination and inhibits integrative recombination. The mechanism of Xis inhibition of the integration reaction was investigated by methylation protection assays (footprinting analyses) in conjunction with recombination assays. Xis is shown to mediate the formation of a specific attP looped structure involving cooperative and competitive long-range interactions among integrase, integration host factor, and Xis proteins. This higher-order structure precludes supercoiled attP from engaging in the productive partner interactions that lead to execution of the first strand exchange in integrative recombination. In addition to its previously characterized role in excision, Xis-induced DNA bending is postulated to act as a regulatory switch (in an alternative loop mechanism) that converts the attP intasome from an integrative-competent complex to a nonreactive one.


Subject(s)
Bacteriophage lambda/genetics , DNA, Viral/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Recombination, Genetic , Viral Proteins , Bacterial Proteins/metabolism , Base Sequence , Carrier Proteins/metabolism , DNA Nucleotidyltransferases/metabolism , DNA-Binding Proteins/metabolism , Integration Host Factors , Kinetics , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids
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