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11.
The tyrosine at position 60 of the Flp recombinase of the Saccharomyces cerevisiae plasmid, 2 mu circle, is invariant among site-specific recombinases of the "yeast plasmid family". Alterations of this residue give rise to Flp variants that show no recombination activity when assayed in vivo in Escherichia coli. Upon purification, they bind substrate, execute DNA cleavage and catalyze recombination. The efficiency of strand cleavage follows the order: Flp(Y60F) greater than Flp greater than Flp(Y60S) greater than Flp(Y60D); efficiency of recombination between Flp sites on a linear substrate and a circular one follows the order: Flp greater than Flp(Y60F) greater than Flp(Y60S) greater than Flp(Y60D). Methylation footprints of the DNA-protein complexes formed by two of the Flp variants, Flp(Y60S) and Flp(Y60D), do not show hypermethylation of the G residues within the substrate core that is characteristic of complexes formed by wild-type Flp. The third variant, Flp(Y60F), causes significant distortion (although less than wild-type Flp) of the substrate core, as indicated by enhanced G-methylation. Binding profiles with circularly permuted substrates indicate that Flp(Y60S) and Flp(Y60D), but not Flp(Y60F), are defective in bending substrate DNA. In recombination between two Flp half-sites, the variant proteins are significantly more active than in normal full-site recombination. 相似文献
12.
The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp. 相似文献
13.
The Flp recombinase of Saccharomyces cerevisae can mediate strand transfer within a half-site, between two half-sites and between a half-site and a full-site. The ability of "step-arrest" mutants of Flp to partake in half-site reactions has been examined. Arg308 variants of Flp, which show little or no strand cleavage in reactions with normal full-sites, execute significant levels of strand transfer in half-site reactions. On the other hand, His305 variants of Flp, which normally accumulate the strand cleavage product from full-sites but do not complete strand transfer, yield only minute amounts of strand transfer products from half-sites. As would be predicted, the step-arrest mutants are unable to produce "normal" or "reverse" recombinants between a half-site and a full-site. The Flp protein is able to form higher-order complexes in association with a half-site. The step-arrest mutants of Flp show specific defects in forming these complexes. 相似文献
14.
DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands. 总被引:9,自引:0,他引:9
Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants. 相似文献
15.
Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis 总被引:16,自引:0,他引:16
The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction. 相似文献
16.
17.
An efficient protocol is described for rapid in vitro multiplication of the vulnerable medicinal herb Drosera indica L. by enhanced axillary bud proliferation from shoot tips as explants. In order to standardize in vitro multiplication of
D. indica, the effects of different strengths of Murashige and Skoog (MS) medium (1/4, 1/3, 1/2 and full strength), different percentages
of sucrose (1, 2 and 3%), various pH (3.7, 4.7, 5.7 and 6.7) and MS basal medium fortified with different concentrations of
zeatin (Z), kinetin (KN) (0.1, 0.5, 1.0 and 2.0 mg/l) and 6-benzylaminopurine (BA) (0.01, 0.05 and 0.1 mg/l) were tried. Multiple
shoot production was independent of different strengths of MS, various percentages of sucrose and also when pH was altered.
Although the number of multiple shoots developed on MS medium supplemented with Z (0.1, 0.5, 1.0 and 2.0 mg/l), KN (0.5 and
1.0 mg/l) and BA (0.1 mg/l) separately was high, the maximum number was observed on MS fortified with Z (0.5 mg/l) and KN
(0.5 mg/l), respectively, which clearly depicts that there is not much difference comparatively with a variation in hormone
concentration in case of Z. High cytokinin concentrations resulted in retardation of shoot growth. Rooting was best achieved
on MS basal medium. This protocol could be useful for production of large biomass within 6 weeks for plumbagin bioprospection
and long term in vitro conservation. 相似文献
18.
Lisby M Olesen JR Skouboe C Krogh BO Straub T Boege F Velmurugan S Martensen PM Andersen AH Jayaram M Westergaard O Knudsen BR 《The Journal of biological chemistry》2001,276(23):20220-20227
All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme. 相似文献
19.
The high copy yeast plasmid 2 microm circle, like the well-studied low copy bacterial plasmids, utilizes two partitioning proteins and a cis-acting 'centromere'-like sequence for its stable propagation. Functionally, though, the protein and DNA constituents of the two partitioning systems are quite distinct. Key events in the yeast and bacterial segregation pathways are plasmid organization, localization, replication, 'counting' of replicated molecules and their distribution to daughter cells. We suggest that the two systems facilitate these common logistical steps by adapting to the physical, biochemical, and mechanical contexts in which the host chromosomes segregate. 相似文献
20.
Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry 总被引:6,自引:0,他引:6
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Tsamis F Gavrilov S Kajumo F Seibert C Kuhmann S Ketas T Trkola A Palani A Clader JW Tagat JR McCombie S Baroudy B Moore JP Sakmar TP Dragic T 《Journal of virology》2003,77(9):5201-5208
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5. 相似文献