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P Modrich

Publications and source records attributed to P Modrich.

At least 55 records · Page 3Linked to original sources
PubMed1982

Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonuclease . DNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.

Base Sequence↗
PubMed1982

Studies on sequence recognition by type II restriction and modification enzymes.

Type II DNA restriction and modification systems are ideally suited for analysis of mechanisms by which proteins specifically recognize unique DNA sequences. Each system is comprised of a unique DNA recognition site and two enzymes, which in those cases examined in detail, are comprised of distinct polypeptide chains. Thus, not only are the DNA substrates extremely well defined, but each system affords the opportunity to compare distinct proteins which interact with a common DNA sequence. This review will focus only on those Type II systems which have been examined in sufficient molecular detail to permit some insight into modes of specific DNA-protein interaction.

Bacteria↗
PubMed1981

DNA determinants important in sequence recognition by Eco RI endonuclease.

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.

Base Sequence↗
PubMed1981

Partial NH2- and cooh-terminal sequence analyses of Eco RI DNA restriction and modification enzymes.

NH2- and COOH-terminal amino acid sequences of the Eco RI restriction and modification enzymes have been determined. The results allow localization of the coding regions within the DNA segment which controls activity of both enzymes. Processing of the endonuclease is limited to removal of NH2-terminal formylmethionine whereas, in the case of the methylase, formylMet-Ala is removed.

Amino Acid Sequence↗
PubMed1980

Membrane phospholipid synthesis in Escherichia coli. Cloning of a structural gene (plsB) of the sn-glycerol-3-phosphate acyl/transferase.

Si+ hybrid ColE1 plasmids of the Clarke-Carbon collection (Clarke, C., and Carbon, J. (1976) Cell 9, 91-99) which eliminate the sn-glycerol 3-phosphate growth requirement of a mutant of Escherichia coli with a Km defect in sn-glycerol-3-phosphate acyltransferase (plsB) were identified. Marked overproduction of a plasmid-encoded sn-glycerol-3-phosphate acyltransferase with a wild type Km in a host plsB- background indicates that the hybrid plasmids carry a structural gene for this enzyme. In addition, all of these plasmids suppress the phenotype of a mutation in a second locus involved in phospholipid biosynthesis, dgk (diglyceride kinase), and one of them also bears the dnaB structural gene. Diglyceride kinase activity is also overproduced in these strains. The linkage of plsB, dgk and dnaB loci was confirmed by transduction analysis which demonstrated the clockwise gene order malB, dnaB, dgk, plsB, and uvrA near Minute 91 on the E. coli linkage map. This is in contrast to the previously reported co-transduction of plsB with dctA near Minute 78 (Cronan, J. E., Jr., and Bell, R. M. (1974) J. Bacteriol., 120, 227-233). Recloning of restriction endonuclease fragments and in vitro mutagenesis have localized the dgk, and plsB loci to a 2.2-megadalton DNA segment, and have demonstrated that diglyceride kinase and sn-glycerol-3-phosphate acyltransferase activities reside in separate polypeptides. Availability of these clones and mutationally altered derivatives has allowed the identification of a single polypeptide (Mr = 83,000) corresponding to the sn-glycerol-3-phosphate acyltransferase and purification of this membrane-bound enzyme to near homogeneity (Larson, T. J., Lightner, V. A., Green, P. R., Modrich, P., and Bell, R. M. (1980) J. Biol. Chem. 255, 9421-9426). The size of the plsB polypeptide indicates that a major fraction of the DNA segment to which this gene has been localized is involved in coding for the sn-glycerol-3-phosphate acyltransferase.

Acyltransferases↗
PubMed1979

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.

DNA-Directed DNA Polymerase↗
PubMed1979

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.

Adenine↗
PubMed1978

Substrate dependence of the mechanism of EcoRI endonuclease.

The mechanism of EcoRI endonuclease is substrate dependent. At 37 degrees dissociation of the enzyme-Form II DNA intermediates of ColE1 DNA and bacteriophage G4 RFI DNA is negligible. Therefore, both DNA strands with in the EcoRI sequence are cleaved during a single binding event. However, double strand cleavage of SV40 DNA occurs without dissociation of the enzyme in only 75% of the catalytic events. This mechanistic difference presumably reflects sequence differences about the EcoRI sites of these DNA's.

Bacteriocin Plasmids↗
PubMed1977

Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes.

The dG residues within the EcoRI recognition sequence of ColE1 DNA have been selectively replaced with dI. Methylation of the altered sequence by the EcoRI modification enzyme is extremely slow as compared with methyl transfer to the natural recognition site. Since the affinity of the modification enzyme for the dI-containing sequence is considerably less than that for the natural sequence, we have concluded that the 2-amino group of dG has an important role in DNA site recognition by this enzyme. In contrast, the altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consisted with our conclusion (Rubin, R. A., and Modrich, P. (1977) J. Biol. Chem. 252, 7265-7272) that the two proteins interact with their common recognition sequence in different ways.

Base Sequence↗
PubMed1976

EcoRI endonuclease. Physical and catalytic properties of the homogenous enzyme.

A procedure for large scale isolation of Escherichia coli RI endonuclease in high yield has been developed. The purified enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and analytical sedimentation. The denatured and reduced form of the enzyme has a molecular weight of 28,500 +/- 500. In solution the enzyme exists as a mixture of dimers and tetramers of molecular weights 57,000 and 114,000, respectively. We estimate the dissociation constant for tetramer to dimer transition to be less than or approximately equal to 1 x 10-7 M. Steady state kinetic analysis of the endonuclease with ColE1 DNA as substrate showed that the enzyme obeys Michaelis-Menten kinetics. At 37 degrees the turnover number is four double strand scissons per min, and the Km for ColE1 molecules is 8 x 10(-9) M. At 0 degrees the major product of endonuclease action contains only one single strand break in the RI site, and such molecules can dissociate from the enzyme. In contrast, at 30 degrees to 37 degrees, two single strand breaks are introduced into the RI sequence prior to dissociation of the enzyme. A transient enzyme-bound intermediate containing only one break in the RI site was observed in studies of a single turnover at 30 degrees. Kinetic analysis of this reaction indicates that the first break is introduced into the RI site with the first order rate constant of at least 40 min-1, while the second cleavage occurs with a rate constant of 14 min-1. Since the turnover number of the enzyme at 30 degress is only 0.72 min-1, these results indicate that the rate-limiting step is release of endonuclear from its DNA product.

Amino Acids↗
PubMed1976

Modification of Escherichia coli DNA ligase by cleavage with trypsin.

Limited treatment of Escherichia coli DNA ligase with trypsin results in rapid loss of DNA joining activity. However, the ability to react with DPN to form the covalent enzyme-AMP intermediate is unaffected. The cleaved enzyme is also unable to catalyze the formation of DNA-adenylate, the second covalent intermediate in the ligase-catalyzed reaction. These findings demonstrate that portions of the DNA ligase molecule that are required for phosphodiester bond formation are not required for at least one of the partial reactions catalyzed by this enzyme.

Escherichia coli↗
PubMed1975

Bacteriophage T7 Deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity.

In vivo, replication of T7 DNA does not occur after infection of Escherchia coli tsnC mutants (CHAMBERLIN, M. (1974) J. Virol. 14, 509-516). In vitro, extracts of tsnC mutant E. coli infected with T7 hage are incapable of replicating duplex T7 DNA, although extracts of wild type E. coli infected with T7 phage support replication of T7 DNA. In addition, extracts of the infected tsnC mutant are deficient in T7 DNA polymerase activity. Extracts prepared from uninfected E.coli tsnC-+ cells restore the ability of the infected tsnC extracts to replicate duplex T7 DNA, and also restore normal levels of the phage DNA polymerase activity. A 12,000-dalton heat-stable protein responsible for this complementation has been purified to near homogeneity from uninfected tsnC+ extracts and it is designated "TsnC protein."

Bacterial Proteins↗