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1.
The integrases are a diverse family of tyrosine recombinases which rearrange DNA duplexes by means of conservative site-specific recombination reactions. Members of this family, of which the well-studied lambda Int protein is the prototype, were previously found to share four strongly conserved residues, including an active site tyrosine directly involved in transesterification. However, few additional sequence similarities were found in the original group of 27 proteins. We have now identified a total of 81 members of the integrase family deposited in the databases. Alignment and comparisons of these sequences combined with an evolutionary analysis aided in identifying broader sequence similarities and clarifying the possible functions of these conserved residues. This analysis showed that members of the family aggregate into subfamilies which are consistent with their biological roles; these subfamilies have significant levels of sequence similarity beyond the four residues previously identified. It was also possible to map the location of conserved residues onto the available crystal structures; most of the conserved residues cluster in the predicted active site cleft. In addition, these results offer clues into an apparent discrepancy between the mechanisms of different subfamilies of integrases. 相似文献
2.
Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex. 相似文献
3.
L Ringrose V Lounnas L Ehrlich F Buchholz R Wade AF Stewart 《Canadian Metallurgical Quarterly》1998,284(2):363-384
The integrase class site specific recombinases FLP from Saccharomyces cerevisiae, and Cre from bacteriophage P1, have been extensively used to direct DNA rearrangements in heterologous organisms. Although their reaction mechanisms have been relatively well characterised, little comparative analysis of the two enzymes has been published. We present a comparative kinetic analysis of FLP and Cre, which identifies important differences. Gel mobility shift assays show that Cre has a higher affinity for its target, loxP (7. 4x10(10) M-1), than FLP for its target, FRT (8.92x10(8) M-1). We show that both recombinases bind the two halves of their target sites cooperatively, and that Cre shows approximately threefold higher cooperativity than FLP. Using a mathematical model describing the sequential binding of recombinase monomers to DNA, we have determined values for the association and dissociation rate constants for FLP and Cre.FLP and Cre also showed different characteristics in in vitro recombination assays. In particular, approximately tenfold more active FLP was required than Cre to optimally recombine a given quantity of excision substrate. FLP was able to reach maximum excision levels approaching 100%, whilst Cre-mediated excision did not exceed 75%. To investigate possible reasons for these differences a mathematical model describing the excision recombination reaction was established. Using measured DNA binding parameters for FLP and Cre in the model, and comparing simulated and experimental recombination data, the values of the remaining unknown parameters were determined. This analysis indicates that the synaptic complex is more stable for Cre than for FLP. 相似文献
4.
We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking. 相似文献
5.
In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model. 相似文献
6.
A new family of explicit pseudodynamic algorithms is proposed for general pseudodynamic testing. One particular subfamily seems very promising for use in general pseudodynamic testing since the stability problem for a structure does not need to be considered. This is because this subfamily is unconditionally stable for any instantaneous stiffness softening system, linear elastic system and instantaneous stiffness hardening system that might occur in the pseudodynamic testing of a real structure. In addition, it also offers good accuracy when compared to a general second-order accurate method for both linear elastic and nonlinear systems. 相似文献
7.
In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells. 相似文献
8.
A Seiler R Reinhardt J Sarkari DA Caugant M Achtman 《Canadian Metallurgical Quarterly》1996,19(4):841-856
The opc gene is widespread in epidemic and endemic Neisseria meningitidis, but most strains of certain epidemic clones (ET-37 complex, Cluster A4) and a few random endemic isolates lack an opc gene. Four percent of the 1148 bp that contain opc plus the surrounding intergenic region was polymorphic (18 alleles), and many of the alleles contained a 230 bp insertion at a fixed location in the intergenic region. The presence or absence of the insertion reflects site-specific recombination. The alleles are stably inherited within clonal groupings for up to at least 50 years, with rare cases of horizontal genetic exchange. Most statistical methods indicated significant intragenic recombination events within this dataset. 相似文献
9.
Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions. 相似文献
10.
We present three experiments which serve to identify carbon and proton sidechain resonances in 13C-labeled proteins. The first is an improvement on the previously published H(C)CH-COSY experiment and comprises the application of gradients for coherence selection and a reduction in the phase cycle. The second experiment is a new (H)CCH-COSY with two carbon dimensions. The (H)CCH-COSY presents several advantages over the H(C)CH-COSY experiment in terms of better sensitivity, improved resolution and easier identification of amino acid spins systems. The third experiment is a 2D proton-edited (H)C(C)H-COSY that allows suppression of methylene resonances. All three HCCH-COSY experiments show good sensitivity and excellent solvent suppression. The 2D version can be acquired in as little as 45 minutes and the 3D versions acquired overnight. The experiments are demonstrated on a 13C-labeled sample of the second PDZ domain from human phosphatase PTP1E in H2O solution. 相似文献
11.
Mechanistic and structural complexity in the site-specific recombination pathways of Int and FLP 总被引:1,自引:0,他引:1
A Landy 《Canadian Metallurgical Quarterly》1993,3(5):699-707
This review focuses on two of the approximately 30 members of the diverse Int family of site-specific recombinases. The lambda recombination system represents those reactions involving accessory proteins and a complex higher-order structure. The FLP system represents the most streamlined reactions and has been the subject of detailed and informative studies on the mechanisms of DNA cleavage and ligation. 相似文献
12.
Various research designs employed in developmental psychology for the investigation of maturational and aging effects are examined. Discrepancies and contradictions in the conclusions derived from cross-sectional and longitudinal studies are consequences of the violation of assumptions implicit in these research designs. The conventional methods are shown to be special cases of a general model for research on behavior change over time. The properties of the general model are explicated and the assumptions for the customary designs are reviewed in the light of these properties. The complete model requires consideration of the components of age, time and cohort differences in the identification of developmental change. Both the longitudinal and cross-sectional methods in this context require strong assumptions which can rarely be met. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
13.
VB Zhurkin G Raghunathan NB Ulyanov RD Camerini-Otero RL Jernigan 《Canadian Metallurgical Quarterly》1994,239(2):181-200
Parallel DNA triplexes considered to be putative intermediates in homologous recombination, are studied by means of theoretical conformational analysis. These triplexes are denoted as the R-form DNA. Two types of triplexes are analyzed: extended R-form DNA, modeling the triple standard structure, created transiently in the presence of recombination proteins (e.g. RecA); and collapsed R-form, obtained after deproteinization. These structures are stereochemically possible for any arbitrary sequence and have the following properties: (1) the third, R-strand, is parallel to the identical duplex strand and is located in the major groove of the duplex; (2) positions of all four bases in the R-strand are nearly isomorphic; (3) the proposed triplets are consistent with the chemical modification data for deproteinized DNA; we suggest, however, that they are the same in the RecA-DNA complex as well. Since the patterns of charges on each base of the R-strand are strictly complementary to the charges of the homologous Watson-Crick (WC) pair in the major groove, we propose that the selection of the homologous sequence may occur through these complementary electrostatic interactions (electrostatic recognition code). We demonstrate that in the collapsed triplex with a rise of about 3.4 A, the bases from the third R-strand can be inclined and interact with two WC base-pairs simultaneously, which could lead to recognition errors. These mispairings are unlikely in the extended triplex. Therefore, we speculate that a functional role of the extended and underwound DNA structure, transiently formed in the complex with RecA protein, is to obviate such errors and increase the stringency of recognition. In other words, RecA plays the role of a DNA chaperone facilitating the recognition of the single stranded DNA and the duplex. Finally, we show that the proposed isomorphic triplets are conformationally advantageous for strand exchange. 相似文献
14.
15.
The site-specific recombinase Intl1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron-associated attl1 site and the 59-base element (59-be) family of sites that are associated with gene cassettes. Intl1 includes the four conserved amino acids that are characteristic of members of the integrase family, and Intl1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. Intl1 was purified as a fusion protein and shown to bind to isolated attl1 or 59-be recombination sites. Binding to attl1 was considerably stronger than to a 59-be. Binding adjacent to the recombination cross-over point was not detected. A strong Intl1 binding site within attl1 was localized by both deletion and footprinting analysis to a 14 bp region 24-37 bp to the left of the recombination cross-over point, and this region is known to be critical for recombination in vivo (Recchia et al., 1994). An imperfect (13/15) direct repeat of this region, located 41-55 bp to the left of the recombination cross-over point, contains a weaker Intl1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding. 相似文献
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17.
A psychologist in general family practice functions as a primary care provider, who is similar to the traditional family doctor who provides treatment for clients and their families. The psychologist has an ongoing interaction with the family and serves as a resource for the family to consult when in difficulty; he or she uses knowledge of the client, as well as knowledge of the client's family, community, and social environment. This approach seems particularly suitable for Asian Americans whose culture emphasizes the role of the family. It helps to minimize the client's inhibition against seeking mental health services and provides the psychologist with certain clinical advantages. Two cases are presented to illustrate the application of this approach to Asian American clients. Situations in which family members should not be seen by the same psychologist and the issues of transference, countertransference, and confidentiality are also considered. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
18.
Contends that the profession of clinical psychology is plagued by problems of role confusion in the public mind, which arises directly from current methods of service delivery. Present psychological service delivery methods are analyzed and found to contain several assumptions that contribute to a poor public image. An alternative model of practice, based on continuous involvement with families over time is described. Adaptive and healthy qualities of consumers of service are stressed. (9 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
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20.
The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases. 相似文献