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Homologous recombination events between circular chromosomes, occurring during or after replication, can generate dimers that need to be converted to monomers prior to their segregation at cell division. In Escherichia coli, chromosome dimers are converted to monomers by two paralogous site-specific tyrosine recombinases of the Xer family (XerC/D). The Xer recombinases act at a specific dif site located in the replication termination region, assisted by the cell division protein FtsK. This chromosome resolution system has been predicted in most Bacteria and further characterized for some species. Archaea have circular chromosomes and an active homologous recombination system and should therefore resolve chromosome dimers. Most archaea harbour a single homologue of bacterial XerC/D proteins (XerA), but not of FtsK. Therefore, the role of XerA in chromosome resolution was unclear. Here, we have identified dif-like sites in archaeal genomes by using a combination of modeling and comparative genomics approaches. These sites are systematically located in replication termination regions. We validated our in silico prediction by showing that the XerA protein of Pyrococcus abyssi specifically recombines plasmids containing the predicted dif site in vitro. In contrast to the bacterial system, XerA can recombine dif sites in the absence of protein partners. Whereas Archaea and Bacteria use a completely different set of proteins for chromosome replication, our data strongly suggest that XerA is most likely used for chromosome resolution in Archaea.

SSV1 is a virus infecting the extremely thermophilic archaeon Sulfolobus shibatae. The viral-encoded integrase is responsible for site-specific integration of SSV1 into its host genome. The recombinant enzyme was expressed in Escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro. We show that the SSV1 integrase belongs to the tyrosine recombinases family and that Tyr 314 is involved in the formation of a 3-phosphotyrosine intermediate. The integrase cleaves both strands of a synthetic substrate in a temperature-dependent reaction, the cleavage efficiency increasing with temperature. A discontinuity was observed in the Arrhenius plot above 50°C, suggesting that a conformational transition may occur in the integrase at this temperature. Analysis of cleavage time course suggested that noncovalent binding of the integrase to its substrate is rate-limiting in the cleavage reaction. The cleavage positions were localized on each side of the anticodon loop of the tRNA gene where SSV1 integration takes place. Finally, the SSV1 integrase is able to cut substrates harboring mismatches in the binding site. For the cleavage step, the chemical nature of the base in position ؊1 of cleavage seems to be more important than its pairing to the opposite strand.Site-specific recombination catalyzed by tyrosine recombinases plays a number of critical roles in prokaryote and eukaryote kingdoms. Well documented examples in lower eukaryotes and bacteria include generation of genetic variability, plasmid copy control and/or stable inheritance, resolution of bacterial chromosome dimers or viral DNA integration in host chromosomes (for reviews, see Refs. 1-3). Members of the tyrosine recombinases family catalyze site-specific recombination between two DNA sites by using a topoisomerase IB-like mechanism to cut and religate DNA strands (4, 5). Unlike topoisomerases, tyrosine recombinases perform the ligation step after strand exchange between the two DNA partners. Sitespecific recombination requires the assembly of a synaptic complex containing, at least, four enzyme protomers and the two DNA sites. The recombination reaction occurs by cutting and exchanging the two pairs of DNA strands in two temporally distinct steps. In the first step, cleavage occurs on the top strands of each DNA site. For each site, a 3Ј-phosphotyrosine DNA-protein covalent complex is formed and a free 5Ј-OH DNA end generated. The leaving strands then attack the phosphotyrosine link of the recombination partner, thus releasing the recombinase subunits. After this first round of strand cleavagestrand exchange, a Holliday junction is formed. The bottom strands are then cut and religated, thus resolving the Holliday junction and completing the recombination reaction.Site-specific recombination in archaea is not as well known. So far, the only studied system is SSV1, a virus of the extremely thermophilic archaeon Sulfolobus shibatae. In the cell, the 15.5-kb genome of SSV1 is present both as a circular DNA and as a provirus stably...

Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding.Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

The only tyrosine recombinase so far studied in archaea, the SSV1 integrase, harbors several changes in the canonical residues forming the catalytic pocket of this family of recombinases. This raised the possibility of a different mechanism for archaeal tyrosine recombinase. The residues of Int SSV tentatively involved in catalysis were modified by site-directed mutagenesis, and the properties of the corresponding mutants were studied. The results show that all of the targeted residues are important for activity, suggesting that the archaeal integrase uses a mechanism similar to that of bacterial or eukaryotic tyrosine recombinases. In addition, we show that Int SSV exhibits a type IB topoisomerase activity because it is able to relax both positive and negative supercoils. Interestingly, in vitro complementation experiments between the inactive integrase mutant Y314F and all other inactive mutants restore in all cases enzymatic activity. This suggests that, as for the yeast Flp recombinase, the active site is assembled by the interaction of the tyrosine from one monomer with the other residues from another monomer. The shared active site paradigm of the eukaryotic Flp protein may therefore be extended to the archaeal tyrosine recombinase Int SSV .Tyrosine recombinases form a large family of site-specific recombinases comprising more than 150 members, most of which were identified on the basis of sequence similarities (1, 2). Within this family, several subfamilies can be defined such as the -phage integrase family, the Xer recombinases family, or the yeast plasmid recombinases family (1). The hallmark of tyrosine recombinases is the conservation of six noncontiguous residues: Arg I , Lys ␤ , His II , Arg II , His/Trp, Tyr (Table I). This motif is directly involved in catalysis of DNA strand cleavage and strand exchange (for review, see Ref.3). Five of the six residues are located within the highly conserved boxes I and II found in tyrosine recombinases (1, 4, 5), whereas the sixth residue, Lys ␤ , was identified by alignments with the eukaryotic topoisomerases IB (6). Two different structural organizations of this motif have been described from crystallographic data. In prokaryotic tyrosine recombinases XerD (7), Cre (8), HP1 integrase (9), and -Int (10, 11), the six active site residues come from a single monomer, whereas the eukaryotic Flp recombinase presents a shared active site, where the catalytic tyrosine is provided by one monomer, and the five other residues are from another monomer (12). In this latter case, the active site is created by dimer association. As a consequence of this organization, Flp realizes trans cleavage (13, 14), whereas prokaryotic recombinases act in cis (8,9,(15)(16)(17). Cis cleavage is the result of cis activation/cis cleavage where the tyrosine of the bound monomer attacks the nearby activated phosphate. In trans cleavage, binding of a monomer to its site leads to activation of the adjacent phosphodiester that will be attacked by a nucleophile (here a tyrosine) provided in tran...

Inspection of the primary sequence of the IS1 transposase suggested that it carries residues which are characteristic of the active site of integrases of the bacteriophage family (Int). In particular, these include a highly conserved triad: His-Arg-Tyr. The properties of mutants made at each of these positions were investigated in vivo. The results of several different assays confirm that each is important for transposase activity. Moreover, as in the case of members of the Int family, different mutations of the His residue exhibited different effects. In particular, a His-to-Leu mutation resulted in complete inactivation whereas the equivalent His-to-Gln mutation retained low but significant levels of activity.

The transposase (InsAB) of the insertion element IS1 can create breaks in DNA that lead to induction of the SOS response. We have used the SOS response to InsAB to screen for host mutations that affect InsAB function and thus point to host functions that contribute to the IS1 transposition mechanism. Mutations in the hns gene, which codes for a DNA binding protein with wide-ranging effects on gene expression, abolish the InsAB-induced SOS response. They also reduce transposition, whether by simple insertion or cointegrate formation, at least 100-fold compared with the frequency seen in hns ؉ cells. Examination of protein profiles revealed that in an hns-null mutant, InsAB is undetectable under conditions where it constitutes the most abundant protein in hns ؉ cells. Likewise, brief labeling of the hns cells with [ 35 S]methionine revealed very small amounts of InsAB, and this was undetectable after a short chase. Transcription from the promoters used to express insAB was essentially unaltered in hns cells, as was the level of insAB mRNA. A mutation in lon, but not in ftsH or clpP, restored InsAB synthesis in the hns strain, and a mutation in ssrA partially restored it, implying that the absence of H-NS leads to a problem in completing translation of insAB mRNA and/or degradation of nascent InsAB protein.

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