Donor Cleavage Research Articles

DNA repair by the cryptic endonuclease activity of Mu transposase

Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835-3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or "5'flap" after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway.

Effects of Donor Cell Passage, Size and Type on Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of cumulus donor cells compared to the >20 cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with 20 donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.

Evidence That HIV-1 Reverse Transcriptase Employs the DNA 3′ End-directed Primary/Secondary RNase H Cleavage Mechanism during Synthesis and Strand Transfer

We previously analyzed strand transfers catalyzed by human immunodeficiency virus, type 1 reverse transcriptase (RT) in a hairpin-containing RNA template system. In this system, RT produces a series of adjacent RNase H cuts before the hairpin base on the first, or donor template that clears a region of the donor, facilitating invasion by the second, or acceptor RNA. Here we analyze characteristics of the prominent cuts before the hairpin base and their role in strand transfers. Analysis of the template cleavage pattern during synthesis suggested that the RT performs DNA 3' end-directed primary and secondary cuts while paused at the hairpin base and that these cuts contribute to creation of the invasion site. RT catalyzed similar cleavages on a substrate representing a paused cDNA-template intermediate. DNA 3' end-directed secondary cuts, which require positioning of the polymerase active site downstream of the primer terminus, had previously not been specifically identified during synthesis. Our findings indicate that during synthesis DNA 3' end-directed primary and secondary cuts occur at pause sites. RT mutants with substitutions at the His(539) residue in the RNase H active site were defective in secondary cleavages. Analysis of the template cleavage pattern generated by the His(539) mutants during synthesis revealed inefficient cleavage at the invasion site, correlating with defects in strand transfer. Overall, results indicate RT can catalyze pause-associated DNA 3' end-directed primary and secondary cuts during synthesis and these cuts can contribute to strand transfer by creation of an invasion site.

Effects of Donor and Acceptor RNA Structures on the Mechanism of Strand Transfer by HIV-1 Reverse Transcriptase

Template switching during reverse transcription contributes to recombination in human immunodeficiency virus type 1 (HIV-1). Our recent studies suggest that the process can occur through a multi-step mechanism involving RNase H cleavage, acceptor invasion, branch migration, and finally primer terminus transfer. In this study, we analyzed the effects of reverse transcriptase (RT)-pausing, RNase H cleavages and template structure on the transfer process. We designed a series of donor and acceptor template pairs with either minimal pause sites or with pause sites at various locations along the template. Restriction sites within the region of homology allowed efficient mapping of the location of primer terminus transfer. Blocking oligomers were used to probe the acceptor invasion site. Introduction of strong pause sites in the donor increased transfer efficiency. However, the new pauses were not necessarily associated with effective invasion. In this system, the primary invasion occurred at a region of donor cleavage associated with weak pausing. These results together with acceptor structure predictions indicated that a potential invasion site is used only in conjunction with a favorable acceptor structure. Stabilizing acceptor structure at the predicted invasion region lowered the transfer efficiency, supporting this conclusion. Differing from previous studies, terminus transfer occurred at a short distance from the invasion site. Introduction of structure into the acceptor template shifted the location of terminus transfer. Nucleocapsid protein, which can improve cDNA-acceptor interactions, increased transfer efficiency with some shift of terminus transfer closer to the invasion site. Overall results support that the acceptor structure has a major influence on the efficiency and position of the invasion and terminus transfer steps.

819. Hyperactive Transposase Mutants of the Sleeping Beauty Transposon

The Sleeping Beauty (SB) transposon system mediates stable genomic integration by a cut-and-paste transposition process and has shown tremendous success as a therapeutic vehicle for in vivo gene delivery. Nevertheless, the overall rate of transposition with SB is still insufficient for many clinical applications. Herein, we generated 141 missense mutants for the SB transposase and screened each for altered gene transfer activity in human cells using a genetic assay. We show that despite equivalent steady-state levels of wild-type (WT) and mutant transposases in transfected cells, many mutations introduced into the N-terminal DNA-binding domain (n=10) and C-terminal catalytic core domain (n=6) resulted in 2- to 4-fold higher transpositional efficiencies compared to WT. In order to better understand the transposition reaction for potential future development, we compared the functional activities of these hyperactive recombinases to that of the WT transposase using a combination of PCR and EMSAs. Results of these studies identified a number of pathways that contribute to improved transposase activity, including an altered affinity of the transposase for transposon ends, transposase conformational changes, enhanced donor cleavage activity, and increased rates of strand transfer. Importantly, many of these hyperactive SB (HSB) mutations functioned synergistically and, when combined with a hyperactive transposon, elevated transposition by 14-fold compared to the first-generation transposon system. Moreover, although WT and mutant transposases were negatively regulated at high enzyme concentrations, the HSB variants supported ~10-fold higher transpositional activity at all experimental doses, suggesting that lower doses could be administered to further improve the safety profile for SB in vivo. In addition, the use of HSB instead of the WT transposase dramatically improved the integration frequency of larger-sized elements, including ones greater than 14 kb in length. These HSB mutants should greatly extend the carrying capacity for SB, thus overcoming one of this system's major limitations. Finally, we studied the long-term in vivo persistence of two transposon reporters, β-galactosidase and human coagulation factor IX (hFIX), in the livers of adult mice following a single administration of WT- or hyperactive transposase-expressing plasmids. Results of these analyses demonstrated that these mutated enzymes could also greatly enhance SB's gene transfer capabilities in vivo, supporting stable integration in an estimated 57% of transfected mouse hepatocytes, compared to ~8% for the WT. This level of transposition was sufficient to maintain ~7-fold higher serum hFIX levels (955 ng/ml ± 241 ng/ml) than achieved in SB-expressing animals (140 ng/ml ± 56 ng/ml) for a period of 4 months (length of study). This level of hFIX obtained with the HSB mutant is ~19% of normal human levels and is well within a curative range capable of converting a severely affected hemophilia B patient to one with a much milder phenotype. Therefore, our work demonstrates the potential for improved in vivo gene delivery through directed transposase evolution, and provides a number of important insights into Sleeping Beauty transposon biology that could be exploited for future development.

Ordered DNA release and target capture in RAG transposition.

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.

Does the proposed DSE motif form the active center in the Hermes transposase?

Donor cleavage and strand transfer are two functions performed by transposases during transposition of class II transposable elements. Within transposable elements, the only active center described, to date, facilitating both functions, is the so-called DDE motif. A second motif, R-K-H/K-R-H/W-Y, is found in the site-specific recombinases of the tyrosine recombinase family. While present in many bacterial insertion sequences as well as in the eukaryotic family of mariner/Tc1 elements, the DDE motif was considered absent in other classes of eukaryotic class II elements such as P, and hAT and piggyBac. Based on sequence alignments of a hobo-like element from the nematode Caenorhabditiselegans, to a variety of other hAT transposases and several members of the mariner/Tc1 group, Bigot et al. [Gene 174 (1996) 265] proposed the presence of a DSE motif in hAT transposases. In the present study we tested if each of these three residues is required for transposition of the Hermes element, a member of the hAT family commonly used for insect transformation. While D402N and E572Q mutations lead to knock-out of Hermes function, mutations S535A and S535D did not affect transposition frequency or the choice of integration sites. These data give the first experimental support that D402 and E572 are indeed required for transposition of Hermes. Furthermore, this study indicates that the active center of the Hermes transposase differs from the proposed DSE motif. It remains to be shown if other residues also form the active site of this transposase.

Mechanisms of Metal Ion Action in Tn10 Transposition

Tn10/IS10 transposition involves assembly of a synaptic complex (or transpososome) in which two transposon ends are paired, followed by four distinct chemical steps at each transposon end. The chemical steps are dependent on the presence of a suitable divalent metal cation (Me 2+). Transpososome assembly and structure are also affected by Me 2+. To gain further insight into the mechanisms of Me 2+ action in Tn10/IS10 transposition we have investigated the effects of substituting Mn 2+ for Mg 2+, the physiologic Me 2+, in transposition. We have also investigated the significance of an Me 2+-assisted conformational change in transpososome structure. We show that Mn 2+ has two previously unrecognized effects on the Tn10 donor cleavage reaction. It accelerates the rates of hairpin formation and hairpin resolution without significantly affecting the rate of the first chemical step, first strand nicking. Mn 2+ also relaxes the specificity of first strand nicking. We also show that Me 2+-assisted transpososome unfolding coincides with a structural transition in the transposon–donor junction that may be necessary for hairpin formation. Possible mechanisms for these observations are considered.

RAG transposase can capture and commit to target DNA before or after donor cleavage.

The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively "safe" regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.

Disruption of target DNA binding in Mu DNA transposition by alteration of position 99 in the Mu B protein

Target DNA binding by the Mu B protein is an important step in phage Mu transposition; however, the region of Mu B involved in target binding and the mechanism of the interaction are unknown. Previous studies have demonstrated that modification of Mu B with the sulfhydryl-specific reagent N-ethylmaleimide can selectively inhibit target DNA binding. We now show that individual mutation of the three cysteines in Mu B to serine results in proteins which are active in intermolecular strand transfer, but demonstrate variable levels of N-ethylmaleimide resistance. The data indicate that cysteine 99 is the primary site of modification affecting target DNA binding, with a minor contribution resulting from the derivitization of cysteine 129. These findings are confirmed by the construction of Mu B mutants containing a bulky side-chain at the individual cysteine to mimic the N-ethylmaleimide modified protein. The C99Y protein shows a complete loss in target-dependent strand transfer activity under standard reaction conditions and C129Y displays partial activity. The effect of the tyrosine substitutions is specific for target interaction as both mutants show wild-type activity in their ability to stimulate the Mu transposase to perform donor cleavage and intramolecular strand transfer. Finally, a target dissociation assay has shown that the C99Y-DNA complex generated in the presence of ATP-γ-S has a drastically reduced half-life as previously found for N-ethylmaleimide treated wild-type Mu B. Modification of cysteine 99 is proposed to block target DNA binding by causing steric interference near the DNA binding pocket.

The mu transposase tetramer is inactive in unassisted strand transfer: an auto-allosteric effect of mu A promotes the reaction in the absence of mu B

A tetramer of the Mu transposase is the structural and functional core in all three stable higher-order nucleoprotein complexes (Type 0, Type 1 and Type 2 transpososomes) generated in a defined in vitro strand transfer reaction. Although functional in donor cleavage, we report here that contrary to previous belief, the Mu A tetramer is incapable of unassisted strand transfer. The Mu B protein is required to stimulate the tetramer for intermolecular strand transfer. In the absence of Mu B protein we show that additional Mu A molecules must be added to the core tetramer to stimulate intramolecular strand transfer. Mapping experiments indicate that domain II of the assisting Mu A mediates functional interactions with the core tetramer. The recipient site for Mu A stimulated strand transfer on the A tetramer is likely in domain II and is clearly different from the domain IIIb site used by the Mu B protein. The Mu accessory end binding sites and the Mu enhancer are not required in the Mu A assisted strand transfer, suggesting that helper A molecules in solution can interact with the core tetramer to stimulate the reaction. Finally, we argue that the strand transfer activity and protein sites for target interaction reside within the core tetramer; hence the role of the stimulatory A molecules appears to be limited to that of an auto-allosteric effector.

Isolation and Characterization of IS 10Transposase Separation of Function Mutants: Identification of Amino Acid Residues in Transposase that are Important for Active Site Function and the Stability of Transposition Intermediates

IS 10transposase mediates end pairing, donor cleavage (or excision) and strand transfer reactions in IS 10/Tn 10transposition. Details of how this multi-functional protein mediates these various steps have not been determined. We have used a genetic approach to help define structure – function relationships for IS 10transposase. IS 10transposase mutants that failed to form an excised transposon fragment in vivoand conferred a dominant-negative phenotype were isolated. Mutants with these properties were expected to be defective in catalysis and/or steps preceding donor cleavage. A total of seven mutants were identified, the corresponding proteins were purified and their activities assessed in vitro. Mutant EK292 was found to form wild-type levels of paired end complex, an early intermediate in the reaction, but to be defective in both donor cleavage and strand transfer. This phenotype suggests that E292 comprises part of a catalytic region. Mutants RH119, SF120 and RQ296 formed aberrant paired end complexes, indicating a defect in a step(s) preceding donor cleavage. Mutants WR98, IS101, and AV162 were found to promote donor cleavage in vitrobut not strand transfer. For IS101 and AV162 the defect in strand transfer may be related to instability of excision complexes. Finally, the observation that IS101, RH119 and AV162 were greatly suppressed by Mn 2+suggests that these mutations have an effect on Mg 2+interactions with transposase.

The phage Mu transpososome core: DNA requirements for assembly and function.

The two chemical steps of phage Mu transpositional recombination, donor DNA cleavage and strand transfer, take place within higher order protein-DNA complexes called transpososomes. At the core of these complexes is a tetramer of MuA (the transposase), bound to the two ends of the Mu genome. While transpososome assembly normally requires a number of cofactors, under certain conditions only MuA and a short DNA fragment are required. DNA requirements for this process, as well as the stability and activity of the ensuing complexes, were established. The divalent cation normally required for assembly of the stable complex could be omitted if the substrate was prenicked, if the flanking DNA was very short or if the two flanking strands were non-complementary. The presence of a single nucleotide beyond the Mu genome end on the non-cut strand was critical for transpososome stability. Donor cleavage additionally required at least two flanking nucleotides on the strand to be cleaved. The flanking DNA double helix was destabilized, implying distortion of the DNA near the active site. Although donor cleavage required Mg2+, strand transfer took place in the presence of Ca2+ as well, suggesting a conformational difference in the active site for the two chemical steps.

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).

Division of labor among monomers within the Mu transposase tetramer

A single tetramer of Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Analysis of C-terminal deletion derivatives of MuA reveals that a 30 amino acid region between residues 575 and 605 is critical for these three steps. Although inactive on its own, a deletion protein lacking this region assembles with the wild-type protein. These mixed tetramers carry out donor cleavage but do not promote strand transfer, even when the donor cleavage stage is bypassed. These data suggest that the active center of the transposase is composed of the C-terminus of four MuA monomers; one dimer carries out donor cleavage while all four monomers contribute to strand transfer.