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J Alves

Publications and source records attributed to J Alves.

At least 37 records · Page 2Linked to original sources
PubMed1996

Asp-59 is not important for the catalytic activity of the restriction endonuclease EcoRI.

The amino acid Asp-59 was proposed to be involved in EcoRI catalyzed DNA cleavage (Cheng et al., EMBO J. 13, 3927-35, 1994). We have tested this hypothesis by site directed mutagenesis experiments. The four mutants D59A, D59E, D59G, and D59N bind with similar stability to the specific recognition sequence as wild type EcoRI. The D59E mutant cleaves DNA as fast as the wild type enzyme. Specific activities of the other three mutants are five to tenfold lower. Therefore, we conclude that Asp-59 is not involved in catalysis of the EcoRI restriction endonuclease. Consequences for catalytic mechanisms of EcoRI and other restriction enzymes are discussed.

Amino Acid Sequence↗
PubMed1996

Increased rate of lupus flare during pregnancy and the puerperium: a prospective study of 78 pregnancies.

The objective was to determine whether the frequency of flare in systemic lupus erythematosus (SLE), patients is increased during pregnancy and the puerperium. Seventy-eight pregnancies in 68 SLE patients attending the lupus pregnancy clinic, at St. Thomas' Hospital, during the last 5 yr were included. The pregnancy period and 8 weeks post-delivery were considered. This group was compared with a control group of 50 consecutive, non-pregnant, age-matched SLE patients attending our weekly lupus clinic. Additionally, 43 of the pregnant patients carried on attending the lupus clinic for the year after puerperium, and their course was compared with themselves during pregnancy. SLE activity was assessed using the Lupus Activity Index (LAI) score. An increase > or = 0.26 in the score was considered as a flare of the disease. Pregnancy and control groups were homogeneous for age, race, disease duration and distribution of autoantibodies. Sixty-five per cent of the patients flared during pregnancy and/or the puerperium and 42% flared in the control group (P = 0.015). The rates of flare per patients/month were 0.082 +/- 0.004 for the pregnancy group and 0.039 +/- 0.003 for the control group (P < 0.001). The 43 patients whose course was controlled after the puerperium flared more frequently during pregnancy that thereafter (McNemar test, P = 0.003). The rates of flare per patient/month were 0.093 +/- 0.006 during pregnancy and the puerperium, and 0.049 +/- 0.004 after the puerperium (P = 0.0015). Kidney and central nervous system involvement was not different between the pregnancy and control groups. In terms of frequency of flares, there was no difference in any of the groups between patients taking and not taking steroids. We conclude that SLE tends to flare during pregnancy. Flares are maximal during the second and third trimester and the puerperium. Flares are not more severe than in non-pregnant patients, and most of the flares can be managed conservatively. Prednisolone does not prevent flares.

Adult↗
PubMed1995

Accuracy of the EcoRV restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences.

In order to investigate the accuracy of the EcoRV restriction endonuclease, we have synthesized a set of double-stranded oligodeoxynucleotides comprising the canonical recognition sequence, the 9 star sequences (i.e., sequences deviating by one base pair from the canonical sequence), and the 18 mismatch sequences (i.e. sequences deviating by one base from the canonical sequence). For each individual single strand of all these 28 substrates we have measured the rate of phosphodiester bond cleavage under normal buffer conditions. Double-strand cleavage of star substrates is at least 5 orders of magnitude slower than cleavage of the canonical substrate. In contrast, most of the mismatch substrates are accepted more readily. In the absence of the essential cofactor Mg2+, EcoRV binds weakly but equally to the canonical and degenerate substrates, (i.e., KDiss is in the micromolar range). However, the inactive catalytic site mutant D90A in the presence of Mg2+ binds the canonical substrate 1-2 orders of magnitude better than degenerate substrates. Therefore, the EcoRV endonuclease needs the essential cofactor Mg2+ to create thermodynamic discrimination between degenerate and canonical sites. But the main discrimination is kinetically controlled and takes place during cleavage. While in the canonical substrate both single strands are cleaved with an equal velocity, in all other substrates one single strand is cleaved faster than the other one, resulting in a dissociation of the enzyme from the DNA between the two cuts. In vivo this may lead to a repair of the erroneous cleavage site by DNA ligases.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1995

Site-directed mutagenesis in the catalytic center of the restriction endonuclease EcoRI.

The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV, BamHI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in a similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro90, Asp91, Glu111 and Lys113) in EcoRI to determine their individual roles in catalysis. The replacement of Asp91 and Lys113, respectively, by conservative mutations (Ala91, Asn91, Ala113, Gln113, His113 and Leu113) resulted in a reduction of binding affinity and complete loss of cleavage activity. Only Lys113-->Arg substitution still allows to cleave DNA, albeit with a rate reduced by at least four orders of magnitude. Lys113 seems to stabilize the structure of the wild-type (wt) ENase since all five ENase variants with mutations at this position show a strongly enhanced tendency to aggregate. The Ala and Gln mutants of Glu111 bind the recognition sequence slightly stronger than wt EcoRI and cleave it with a low, but detectable rate. Only the Glu111-->Lys mutant, in which the charge is reversed, shows neither binding nor cleavage activity. Pro90 is not important for catalysis, because the Ala90 mutant cleaves DNA with an only slightly reduced rate. Under star conditions, however, this mutant is even more active than wt EcoRI. Therefore, the charged aa Asp91, Glu111 and Lys113 are essential for catalytic activity of the EcoRI ENase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

A dodecapeptide comprising the extended chain-alpha 4 region of the restriction endonuclease EcoRI specifically binds to the EcoRI recognition site.

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.

Amino Acid Sequence↗
PubMed1995

Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites.

EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity. We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements. The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein. Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules. After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer. The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies. Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII. It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible. We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity.

Base Sequence↗
PubMed1994

Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA.

Linear diffusion is a mechanism to accelerate association rates beyond their three-dimensional diffusional limit. It is employed by the restriction endonuclease EcoRI as well as many other proteins interacting with specific DNA sequences to locate their target sites on the macromolecular substrate. In order to investigate biochemical and biophysical details of the linear diffusion process, we have developed a competitive cleavage assay which allows us to assess with great accuracy the influence of sequence, sequence context, and other structural features on the linear diffusion of EcoRI on DNA. We show here that linear diffusion is not a hopping but a sliding movement in which EcoRI follows the helical pitch of the DNA, because it does not "overlook" any cleavage site. Linear diffusion is slowed when EcoRI encounters sites on the DNA which resemble its recognition site ("star" sites). Pauses of up to 20 s are induced, depending on sequence and orientation of the star site. These data suggest that EcoRI can bind to DNA in two binding modes: one tight, specific, and immobile, leading to DNA cleavage, and another one loose and nonspecific, allowing for linear diffusion. Depending on the similarity between the recognition sequence and the DNA sequence being encountered by EcoRI, there will be a continuous transition between these binding modes. Other proteins bound to the DNA and irregular DNA structures such as bent DNA or a triple helix constitute a barrier that cannot easily be passed by EcoRI.

Base Sequence↗
PubMed1994

[Prevalence of asthma in the city of Porto].

It is possible that asthma epidemiology will contribute to the definition of asthma precipitant factors. This work is enrolled in an international study co-ordinated by COMAC-EPI. In Oporto, among 137.000 residents in the same town region, a standardised sample of 4.047 male and female individuals aged from 20 to 44 years was defined. A screening questionnaire was sent with a stamped envelope. Four months later the same questionnaire was sent to the individuals who did not answer it and finally those who do not answer the second letter were visited in their own residence. We got 2075 answers. Among the responders about half were women: 1075, 25.39% belonging (273) to the 20-29 years age group; 42.69% (459) 30-39 years; 31.90% (343) 40-44 years. In men the percentage of distribution by age groups was similar. Question nQ 5 was answered affirmatively by 60 individuals, corresponding to the prevalence of 2.89%. Seventy-one (3.42%) said they had been under asthma treatment during the last year. Among the symptoms that usually define the attack of asthma, those inquired in the group of question 1 show a prevalence close to that of the asthma diagnosis: 6.45%. The prevalence of being awakened by tightness in the chest, shortness of breath and coughing, with values of 16.17%, 10.69% and 25.68% is quite superior than in question nQ 5. The prevalence obtained with the Hay Fever question was 18.84%. Data obtained is similar to the data of other centers.

Adult↗
PubMed1993

Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes.

The crystal structure analyses of the EcoRI-DNA and EcoRV-DNA complexes do not provide clear suggestions as to which amino acid residues are responsible for the activation of water to carry out the DNA cleavage. Based on molecular modeling, we have proposed recently that the attacking water molecule is activated by the negatively charged pro-Rp phosphoryl oxygen of the phosphate group 3' to the scissile phosphodiester bond. We now present experimental evidence to support this proposal. (i) Oligodeoxynucleotide substrates lacking this phosphate group in one strand are cleaved only in the other strand. (ii) Oligodeoxynucleotide substrates carrying an H-phosphonate substitution at this position in both strands and, therefore, lacking a negatively charged oxygen at this position are cleaved at least four orders of magnitude more slowly than the unmodified substrate. These results are supported by other modification studies: oligodeoxynucleotide substrates with a phosphorothioate substitution at this position in both strands are cleaved only if the negatively charged sulfur is in the RP configuration as shown for EcoRI [Koziolkiewicz, M. & Stec, W.J. (1992) Biochemistry 31, 9460-9466] and EcoRV (B. A. Connolly, personal communication). As the phosphate residue 3' to the scissile phosphodiester bond is not needed for strong DNA binding by both enzymes, these findings strongly suggest that this phosphate group plays an active role during catalysis. This proposal, furthermore, gives a straightforward explanation of why in the EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted differently, but in each case the 3' phosphate group closely approaches the phosphate group that is attacked. Finally, an alternative mechanism for DNA cleavage involving two metal ions is unlikely in the light of our finding that both EcoRI and EcoRV need only one Mg2+ per active site for cleavage.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence -GAATTC- and for coupling specific DNA binding to catalysis.

The Gln115 residue of the EcoRI restriction endonuclease has been proposed to form a hydrophobic contact to the methyl group of the inner thymidine of the EcoRI recognition sequence -GAATTC- and to be involved in intramolecular hydrogen bonds to the mainchain at positions 140 and 143 as well as to the side-chain of Asn173. We have exchanged Gln115 for Ala and Glu by site-directed mutagenesis and analysed the purified mutant proteins (Q115A and Q115E) biochemically and physico-chemically. Q115A and Q115E have the same secondary structure composition as wild-type EcoRI but are less stable towards thermal denaturation than the wild-type enzyme. In contrast to wild-type EcoRI the mutant proteins show a biphasic denaturation profile under alkaline pH, presumably because the amino acid exchange labilizes one part of the molecule, which unfolds before the rest of the protein is denatured. Q115A is catalytically inactive under normal buffer conditions, in part due to a diminished affinity towards DNA. At low ionic strength and alkaline pH, as well as in the presence of Mn2+, i.e. under conditions where wild-type EcoRI shows a relaxed specificity, Q115A is active, however not as much as wild-type EcoRI. Under these conditions it cleaves the canonical sequence -GAATTC- with the same kcat/Km value as the sequence -GAAUTC-, which differs from the former sequence by a single methyl group, while wild-type EcoRI shows a tenfold lower kcat/Km for cleavage of -GAAUTC- than for -GAATTC-. Binding experiments, carried out in the absence of Mg2+, demonstrate that Q115A has a similar affinity towards -GAATTC- as to -GAAUTC-, while wild-type EcoRI binds to -GAATTC- with a tenfold preference over -GAAUTC-. On the basis of these thermodynamic and kinetic results it can be concluded that the hydrophobic contact between the gamma-methylene group of Gln115 and the methyl group of the inner thymidine contributes about 3 kJ/mol (0.7 kcal/mol) to the energy of interaction, both in the ground and the transition state. Q115E is catalytically inactive under normal buffer conditions, but becomes active at low ionic strength or in the presence of Mn2+. Different from Q115A, Q115E is inactive at alkaline pH and its DNA binding affinity is highest at acidic pH.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1993

A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases.

We have developed an assay procedure to monitor the cleavage of DNA substrates by restriction endonucleases. This procedure uses DNA substrates that are labeled with biotin on one 5' end and with an antigenic group, e.g., fluorescein or digoxigenin, on the other 5' end. After incubation with the restriction enzyme, the reaction is stopped with EDTA and an aliquot is pipetted into the well of an avidin-coated microtiter plate. This immobilizes the unreacted substrate and the biotinylated cleavage product, whereas the other cleavage product labeled with the antigenic group is subsequently washed off. The unreacted substrate is detected by an enzyme-linked immunosorbent assay with an appropriate enzyme-linked antibody. To test our assay we have measured the steady-state rate constants for cleavage of DNA by EcoRI yielding a kcat of 8.6 min-1 and a Km of 150 nM, which are close to values measured with other assays. The advantage of this assay is that it is not only fast and accurate, but also very sensitive. It allows for many samples to be analyzed in parallel and lends itself to automation. Furthermore, this assay can be designed as a competitive assay, when two substrates carrying different antigenic groups are used. The usefulness of such competitive assay is demonstrated by determining the influence of sequence context on the rate of DNA cleavage by EcoRI.

Base Sequence↗
PubMed1993

Characterization of two "Metabacterium" sp. from the gut of rodents. 2. Heteroxenic cultivation and proof of dipicolinic acid in "M. polyspora".

The vegetative cell of "Metabacterium polyspora" is "cucumber-shaped", about 21 x 5.7 microns, Gram-negative. Cylindrical endospores are best stained by Rakette and Ziehl-Neelsen staining. The bacterium reproduces by sporulation (2 to 8 endospores per cell) and by binary fission. Lateral, bow-like "hatching" of the endospores was seen. About 52% of guinea pigs harbor 5 x 10(6), 36% below 2 x 10(6) and 1% more than 1 x 10(8) "M. polyspora" in 1 g of caecal content. Dipicolinic acid was demonstrated using HPLC in the caecum homogenate from a guinea pig. The amount of it was proportional to the number of spores. Cultivation under strict anaerobic conditions did not succeed. It was possible to cultivate this giant endosymbiont in vitro in a heteroxenic culture incubated in a 5% CO2 atmosphere using liquid medium supplemented with cell-free filtrate of the caecum. The caecum filtrate containing undefined growth factor(s) is necessary for long-term culture. The replication rate was low. These findings suggest that the giant endosymbiont "M. polyspora" is a spore-forming prokaryote without the attributes of a strict anaerobe.

Animals↗
PubMed1992

On the catalytic mechanism of EcoRI and EcoRV. A detailed proposal based on biochemical results, structural data and molecular modelling.

EcoRI and EcoRV have a very similar active site, as is apparent from a comparison of the structures of their respective protein-DNA complexes. Based on structural and mechanistic data, as well as detailed molecular modelling presented here, a mechanism for the DNA cleavage by these enzymes is suggested in which the attacking water molecule is activated by the phosphate group 3' to the scissile phosphodiester bond, and in which the leaving group is protonated by a water molecule associated with the essential cofactor, Mg2+. The mechanism proposed may also apply to other nucleases.

Binding Sites↗
PubMed1992

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV.

We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.

Amino Acid Sequence↗
PubMed1992

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.

Base Sequence↗
PubMed1990

Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences.

We have synthesized a series of 18 nonpalindromic oligodeoxynucleotides that carry all possible base changes within the recognition sequence of EcoRI. These single strands can be combined with their complementary single strands to obtain all possible EcoRI sequences (left), or they can be combined with a single strand containing the canonical sequence to obtain double strands with all possible mismatches within the recognition sequence (right): (sequence; see text) The rate of phosphodiester bond cleavage of these oligodeoxynucleotides by EcoRI was determined in single-turnover experiments under normal buffer conditions in order to find out to what extent the canonical recognition site can be distorted and yet serve as a substrate for EcoRI. Our results show that oligodeoxynucleotides containing mismatch base pairs are in general more readily attacked by EcoRI than oligodeoxynucleotides containing EcoRI sites and that the rates of cleavage of the two complementary strands of degenerate oligodeoxynucleotides are quite different. We have also determined the affinities of these oligodeoxynucleotides to EcoRI. They are higher for oligodeoxynucleotides carrying a mismatch within the EcoRI recognition site than for oligodeoxynucleotides containing an EcoRI site but otherwise do not correlate with the rate with which these oligodeoxynucleotides are cleaved by EcoRI. Our results allow details to be given for the probability of EcoRI making mistakes in cleaving DNA not only in its recognition sequence but also in sequences closely related to it. Due to the fact that the rates of cleavage in the two strands of a degenerate sequence generally are widely different, these mistakes are most likely not occurring in vivo, since nicked intermediates can be repaired by DNA ligase.

Base Composition↗