Biomedical subjects

S E Nunes-Düby

Publications and source records attributed to S E Nunes-Düby.

11 recordsLinked to original sources

PubMed1998

Similarities and differences among 105 members of the Int family of site-specific recombinases.

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).

Amino Acid Sequence↗

PubMed1997

Sensing homology at the strand-swapping step in lambda excisive recombination.

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.

Bacterial Proteins↗

PubMed1995

Single base-pair precision and structural rigidity in a small IHF-induced DNA loop.

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.

Bacterial Proteins↗

PubMed1995

Swapping DNA strands and sensing homology without branch migration in lambda site-specific recombination.

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.

Bacteriophage lambda↗

PubMed1994

Lambda integrase cleaves DNA in cis.

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.

Bacteriophage lambda↗

PubMed1990

Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL.

To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants. The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination. It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain. This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner.

Bacteriophage lambda↗

PubMed1989

Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination.

The early events in site-specific excisive recombination were studied with phage lambda half-att sites that have no DNA to one side of the strand exchange region; they carry a single core-type integrase binding site and either P or P' arm flanking DNA. These half-attR and half-attL sites exhibit normal properties for the initial (covalent) top-strand transfer and form stable intermediates independent of later steps in the reaction. With these novel substrates we show that Xis specifically promotes the first strand exchange and that attL enhances Int cleavage at the top-strand site of attR. It is also shown that synapsis and initial strand transfers do not require DNA-DNA pairing but are mediated by protein-protein and protein-DNA interactions. These involve the two top-strand Int binding sites (required for the first strand exchange) and, in addition, one of the two bottom-strand sites (C') responsible for the second strand exchange.

Attachment Sites, Microbiological↗

PubMed1988

Suicide recombination substrates yield covalent lambda integrase-DNA complexes and lead to identification of the active site tyrosine.

High levels of covalent integrase-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by lambda integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine-342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the "integrase family." The present results show that this small region of homology participates in catalysis of strand transfer.

Amino Acid Sequence↗

PubMed1987

Site-specific recombination intermediates trapped with suicide substrates.

A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.

Bacteriophage lambda↗

PubMed1986

Structural and regulatory divergence among site-specific recombination genes of lambdoid phage.

The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini.

Amino Acid Sequence↗

PubMed1985

Generation of single base-pair deletions, insertions, and substitutions by a site-specific recombination system.

The sequence analysis of both products of individual phi 80 site-specific recombination events in vivo shows that recombination with a secondary attachment (att) site generates several different novel joints at the mismatched position: one recombination event resulted in a single base-pair deletion and two other recombination events resulted in two different single base-pair substitutions. The characterized products of recombination can be straightforwardly interpreted as the outcome of strand exchange involving staggered nicks bracketing the heterology within an overlap region of five to nine base pairs. In comparison, more complex segregation patterns have been observed in previous studies of lambda recombination between nonidentical att sites; the nature of the overlap region heterology may have a significant effect on the segregation patterns. To recover both products of a single recombination event, we used a plasmid that carries the phi 80 int and xis genes and both att sites. Because the two att sites are situated in opposite orientation, intramolecular recombination between them inverts rather than deletes the intervening segment of DNA. Although subsequent reinversion restores the original gross genetic arrangement, single base-pair insertions, deletions, and substitutions are introduced at the sites of recombination. One of the mutations improves the recombination efficiency of the secondary att site and thereby converts a formerly "stable" sequence to an efficient target for rearrangement, and other mutations are predicted to alter the specificity of recombination. These pathways may also provide useful models for the efficient generation of localized sequence diversity on a development (as well as evolutionary) time scale.

Bacteriophage lambda↗