Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.

The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

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.

Evidence for a Xer/dif system for chromosome resolution in archaea.

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.

Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase.

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.

Origin and evolution of DNA topoisomerases.

The DNA topoisomerases are essential for DNA replication, transcription, recombination, as well as for chromosome compaction and segregation. They may have appeared early during the formation of the modern DNA world. Several families and subfamilies of the two types of DNA topoisomerases (I and II) have been described in the three cellular domains of life (Archaea, Bacteria and Eukarya), as well as in viruses infecting eukaryotes or bacteria. The main families of DNA topoisomerases, Topo IA, Topo IB, Topo IC (Topo V), Topo IIA and Topo IIB (Topo VI) are not homologous, indicating that they originated independently. However, some of them share homologous modules or subunits that were probably recruited independently to produce different topoisomerase activities. The puzzling phylogenetic distribution of the various DNA topoisomerase families and subfamilies cannot be easily reconciled with the classical models of early evolution describing the relationships between the three cellular domains. A possible scenario is based on a Last Universal Common Ancestor (LUCA) with a RNA genome (i.e. without the need for DNA topoisomerases). Different families of DNA topoisomerases (some of them possibly of viral origin) would then have been independently introduced in the different cellular domains. We review here the main characteristics of the different families and subfamilies of DNA topoisomerases in a historical and evolutionary perspective, with the hope to stimulate further works and discussions on the origin and evolution of these fascinating enzymes.

A universal type IA topoisomerase fold.

A class of enzymes, called DNA topoisomerases, is responsible for controlling the topological state of cellular DNA. Among these, type IA topoisomerases form a vast family that is present in all living organisms, including higher eukaryotes, in which they play important roles in genome stability. The known 3D structures of three of these enzymes indicate that they share a common toroidal architecture. We previously showed that the toroidal structure could be split off from the core enzyme of Thermotoga maritima topoisomerase I by limited proteolysis. This structure is produced by the association of two tandemly repeated elementary folds in a head-to-tail orientation. By using a combination of structural and sequence data analysis, we show that the elementary fold of about 150 amino acid residues, referred to as the topofold, is likely to be present in the whole topoisomerase IA family. Within each enzyme, the successive topofolds share two conserved sequence motifs located at the base of the ring, and referred to as the MI and MII motifs. However, the overall sequences of the folds have largely diverged. By contrast, secondary and tertiary structures appear remarkably conserved. We suggest that this twofold repeat has evolved by gene duplication/fusion from an ancestral topofold.

Mutational analysis of the archaeal tyrosine recombinase SSV1 integrase suggests a mechanism of DNA cleavage in trans.

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).

Protective role for H-NS protein in IS1 transposition.

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 [35S]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.

Cleavage properties of an archaeal site-specific recombinase, the SSV1 integrase.

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 degrees 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.