共查询到20条相似文献,搜索用时 15 毫秒
1.
DT Minnick K Bebenek WP Osheroff RM Turner M Astatke L Liu TA Kunkel CM Joyce 《Canadian Metallurgical Quarterly》1999,274(5):3067-3075
To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3'-5'-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzyme-substrate interactions and is not easily perturbed. In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site. Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. We separated the contributions of the nucleotide insertion and mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions. The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T.G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A. 相似文献
2.
Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2'-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 "steric gate" can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides. 相似文献
3.
Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075-3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus. 相似文献
4.
DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primer-templates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present. 相似文献
5.
Recognition and manipulation of branched DNA structure by junction-resolving enzymes 总被引:2,自引:0,他引:2
The junction-resolving enzymes are a class of nucleases that introduce paired cleavages into four-way DNA junctions. They are important in DNA recombination and repair, and are found throughout nature, from eubacteria and their bacteriophages through to higher eukaryotes and their viruses. These enzymes exhibit structure-selective binding to DNA junctions; although cleavage may be more or less sequence-dependent, binding affinity is purely related to the branched structure of the DNA. Binding and cleavage events can be separated for a number of the enzymes by mutagenesis, and mutant proteins that are defective in cleavage while retaining normal junction-selective binding have been isolated. Critical acidic residues have been identified in several resolving enzymes, suggesting a role in the coordination of metal ions that probably deliver the hydrolytic water molecule. The resolving enzymes all bind to junctions in dimeric form, and the subunits introduce independent cleavages within the lifetime of the enzyme-junction complex to ensure resolution of the four-way junction. In addition to recognising the structure of the junction, recent data from four different junction-resolving enzymes indicate that they also manipulate the global structure. In some cases this results in severe distortion of the folded structure of the junction. Understanding the recognition and manipulation of DNA structure by these enzymes is a fascinating challenge in molecular recognition. 相似文献
6.
TS Krishna XP Kong S Gary PM Burgers J Kuriyan 《Canadian Metallurgical Quarterly》1994,79(7):1233-1243
The crystal structure of the processivity factor required by eukaryotic DNA polymerase delta, proliferating cell nuclear antigen (PCNA) from S. cerevisiae, has been determined at 2.3 A resolution. Three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring. The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase. The trimeric PCNA ring is strikingly similar to the dimeric ring formed by the beta subunit (processivity factor) of E. coli DNA polymerase III holoenzyme, with which it shares no significant sequence identity. This structural correspondence further substantiates the mechanistic connection between eukaryotic and prokaryotic DNA replication that has been suggested on biochemical grounds. 相似文献
7.
T Paz-Elizur M Takeshita M Goodman M O'Donnell Z Livneh 《Canadian Metallurgical Quarterly》1996,271(40):24662-24669
Bypass synthesis by DNA polymerase II was studied using a synthetic 40-nucleotide-long gapped duplex DNA containing a site-specific abasic site analog, as a model system for mutagenesis associated with DNA lesions. Bypass synthesis involved a rapid polymerization step terminating opposite the nucleotide preceding the lesion, followed by a slow bypass step. Bypass was found to be dependent on polymerase and dNTP concentrations, on the DNA sequence context, and on the size of the gap. A side-by-side comparison of DNA polymerases I, II, and III core revealed the following. 1) Each of the three DNA polymerases bypassed the abasic site analog unassisted by other proteins. 2) In the presence of physiological-like salt conditions, only DNA polymerase II bypassed the lesion. 3) Bypass by each of the three DNA polymerases increased dramatically in the absence of proofreading. These results support a model (Tomer, G., Cohen-Fix, O. , O'Donnell, M., Goodman, M. and Livneh, Z. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1376-1380) by which the RecA, UmuD, and UmuC proteins are accessory factors rather than being absolutely required for the core mutagenic bypass reaction in induced mutagenesis in Escherichia coli. 相似文献
8.
The effect of intercalating compounds such as 9-aminoacridine, quinacrine (atebrin), proflavine and daunomycin on the activity of DNA polymerase I(EC 2.7.7.7) was studied in vitro and compared with the binding of these acridines to native DNA. The enzyme kinetics were followed at various concentrations of DNA 3'-OH primer end groups and constant concentrations of deoxynucleosidetriphosphates as well as under the opposite conditions. The Km values for the DNA 3'-OH end groups were 16--38 nM and for the deoxynucleosidetriphosphates 2--5 micrometer, depending on the buffer and pH used in the enzymatic assay. All acridine derivates inhibit the DNA polymerase; at variable DNA concentrations a competitive inhibition was observed, where the Ki values ranged between 0.87 and 8.5 micrometer. At variable concentrations of deoxynucleosidetriphosphates and constant DNA concentration a non-competitive inhibition was observed. On denatured 3'-OH DNA as well as on poly(dA) - (dT)10 as substrate no inhibition by 9-aminoacridine was observed. 5'--3' exonuclease activity of DNA polymerase is inhibited by 9-aminoacridine but 3'--5' exonuclease activity on denatured DNA is not influenced by this intercalating compound. The affinity of the acridines to DNA was determined spectrophotometrically under conditions similar to those in the enzymatic assay and the computed frequency of intercalation was related to the inhibition of enzymatic activity. The mechanism of inhibition is explained by a disturbance of the structure of the double helical DNA due to the interaction of the bound acridine derivates. 相似文献
9.
Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques. 相似文献
10.
Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1. 相似文献
11.
JF Davies RJ Almassy Z Hostomska RA Ferre Z Hostomsky 《Canadian Metallurgical Quarterly》1994,76(6):1123-1133
The crystal structure of the catalytic domain of rat DNA polymerase beta (pol beta) has been determined at 2.3 A resolution and refined to an R factor of 0.22. The mixed alpha/beta protein has three subdomains arranged in an overall U shape reminiscent of other polymerase structures. The folding topology of pol beta, however, is unique. Two divalent metals bind near three aspartic acid residues implicated in the catalytic activity. In the presence of Mn2+ and dTTP, interpretable electron density is seen for two metals and the triphosphate, but not the deoxythymidine moiety. The principal interaction of the triphosphate moiety is with the bound divalent metals. 相似文献
12.
EK Dimitriadis R Prasad MK Vaske L Chen AE Tomkinson MS Lewis SH Wilson 《Canadian Metallurgical Quarterly》1998,273(32):20540-20550
The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites for self-association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol beta interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol beta did not. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain. 相似文献
13.
Escherichia coli DNA topoisomerase I is a well-studied type I DNA topoisomerase that catalyzes the breakage and rejoining of one DNA strand to allow passage of the other strand. We have cloned and over-expressed a 67 kDa amino-terminal fragment of the protein, and shown that it retains the ability of the intact enzyme to cleave single-stranded DNA. High-quality crystals of the purified 67 kDa fragment have been obtained. The crystals belong to space group P2(1)2(1)2(1), with cell dimensions a = 64.0 A, b = 79.9 A and c = 142.3 A. They diffract to at least 2.8 A at low temperature and, when cooled to cryogenic temperatures, to at least 1.9 A in a synchrotron source. A complete native data set and two derivative data sets have been collected. A multiple isomorphous replacement map to 3 A resolution shows clear secondary structural elements. Final structure determination is in progress. 相似文献
14.
Effect of cross-linking on the secondary structure of DNA I. Cross-linking by photodimerization 总被引:4,自引:0,他引:4
An investigation has been made into the effect produced by photo-induced pyrimidine cross-links upon the secondary structure of DNA. We have studied the effect of uv irradiation upon the B in equilibrium A transition in DNA brought about by a change of solvent from 70 to 80% ethanol. Circular dichroism (CD) was used to monitor the conformational changes. However, we first showed by means of laser Raman spectroscopy that CD is a reliable monitor of the conformational change, even though the DNA is aggregated in 80% alcohol solutions. It is suggested that this aggregation stabilizes the A form through lateral interaction between the helices. The uv irradiation experiments show that pyrimidine-dimer cross-links induced into the B-form DNA will lock it irreversibly into that conformation and prevent it from going to the A form in 80% EtOH solution. The A-form DNA can tolerate a few cross-links but converts cooperatively to the B form if a larger number of cross-links is introduced. Irradiation-induced pyrimidine cross-links create locally denatured regions in B-form DNA. Upon continued irradiation, the entire DNA moelcule becomes denatured. 相似文献
15.
A Bonnin MN Fourmaux JF Dubremetz RG Nelson P Gobet G Harly M Buisson D Puygauthier-Toubas G Gabriel-Pospisil M Naciri P Camerlynck 《Canadian Metallurgical Quarterly》1996,137(2-3):207-211
In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates. 相似文献
16.
The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%). The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA. The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation. 相似文献
17.
DF Chen I Seibert HY Chen I Herbst-Kiene LT Pastucha W Stangel 《Canadian Metallurgical Quarterly》1998,51(6):645-648
These are hard times for medical school deans--high turnover among deans, the fiscal distress of many medical schools, the gap between what deans expect the job will be and what is required of them, the stark differences between what the job of dean is today and what it was in the past, and the threats to the academic missions of education and research. Using stories, anecdotes, and parables, the authors illustrates how these very difficulties might be an opportunity to rethink the role of deans and to re-examine the attributes and skills required of successful deans today. The ultimate goals of medical education have not changed, but the drastic nature of the changes taking place all around, and within, medical education make it more critical than ever to keep in mind what is really important. Deans must be exquisitely attuned to what is really important and they must make sure that the academic medical community never loses sight of what that is. To do that, deans must be deeply rooted personally in the enduring values and commitments that inform medicine as a profession and a vocation and in the fundamental values of medical education and scholarship; they must personify and embody these values; and they must remind us of these values and inspire us to embrace them and be guided by them. This is the sense in which deans must be "spiritual" leaders--that is, through their personal example, they must rekindle and engage the spirit of those working on behalf of the academic mission. While the need for fiscal expertise, management skills, and diplomatic and interpersonal skills in deans is widely acknowledged, the need for sensitivity to the spiritual dimensions of the work of deans has not received the attention it deserves. 相似文献
18.
CM Simbulan M Suzuki S Izuta T Sakurai E Savoysky K Kojima K Miyahara Y Shizuta S Yoshida 《Canadian Metallurgical Quarterly》1993,268(1):93-99
The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes. 相似文献
19.
Eukaryotic DNA polymerase beta (pol beta) can catalyze DNA synthesis during base excision DNA repair. It is shown here that pol beta also catalyzes release of 5'-terminal deoxyribose phosphate (dRP) residues from incised apurinic-apyrimidinic sites, which are common intermediate products in base excision repair. The catalytic domain for this activity resides within an amino-terminal 8-kilodalton fragment of pol beta, which comprises a distinct structural domain of the enzyme. Magnesium is required for the release of dRP from double-stranded DNA but not from a single-stranded oligonucleotide. Analysis of the released products indicates that the excision reaction occurs by beta-elimination rather than hydrolysis. 相似文献