V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture. (+info)
Monte Carlo implementation of supercoiled double-stranded DNA.
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Metropolis Monte Carlo simulation is used to investigate the elasticity of torsionally stressed double-stranded DNA, in which twist and supercoiling are incorporated as a natural result of base-stacking interaction and backbone bending constrained by hydrogen bonds formed between DNA complementary nucleotide bases. Three evident regimes are found in extension versus torsion and force versus extension plots: a low-force regime in which over- and underwound molecules behave similarly under stretching; an intermediate-force regime in which chirality appears for negatively and positively supercoiled DNA and extension of underwound molecule is insensitive to the supercoiling degree of the polymer; and a large-force regime in which plectonemic DNA is fully converted to extended DNA and supercoiled DNA behaves quite like a torsionless molecule. The striking coincidence between theoretic calculations and recent experimental measurement of torsionally stretched DNA (Strick et al., Science. 271:1835, 1996; Biophys. J. 74:2016, 1998) strongly suggests that the interplay between base-stacking interaction and permanent hydrogen-bond constraint takes an important role in understanding the novel properties of elasticity of supercoiled DNA polymer. (+info)
Sequential strand exchange by XerC and XerD during site-specific recombination at dif.
(59/1746)
Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges. (+info)
Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling.
(60/1746)
Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor. (+info)
RNase H overproduction corrects a defect at the level of transcription elongation during rRNA synthesis in the absence of DNA topoisomerase I in Escherichia coli.
(61/1746)
It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation. (+info)
DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme.
(62/1746)
The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed. (+info)
Site-specific recombination in mammalian cells catalyzed by gammadelta resolvase mutants: implications for the topology of episomal DNA.
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We have transferred the prokaryotic gammadelta resolvase system to mammalian cells and present a comparative analysis of recombination by wild-type and two mutant resolvases (E124Q and E102Y/E124Q). Transient co-transfection assays using beta-galactosidase as reporter for recombination reveal that episomal DNA does not contain a significant level of unconstrained negative supercoiling, since only mutant resolvases are recombination-proficient. We also show that the efficiency of recombination by the resolvase double mutant is comparable to that observed with Cre, which indicates that resolvase can be used as a new tool for controlled manipulations of episomal DNAs. (+info)
Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration.
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The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents. (+info)