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P Modrich

Publications and source records attributed to P Modrich.

At least 19 recordsLinked to original sources
PubMed1992

Initiation of methyl-directed mismatch repair.

Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K. M., Lu, A.-L., Clark, S., and Modrich, P. (1987) J. Biol. Chem. 262, 15624-15629). MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA. The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T greater than G-G greater than A-C greater than C-C), and activated MutH responds to the state of DNA adenine methylation. Incision of an unmethylated strand occurs immediately 5' to a d(GATC) sequence, leaving 5' phosphate and 3' hydroxy termini (pN decreases pGpAp-TpC). Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine. The mechanism of activation apparently requires ATP hydrolysis since adenosine-5'-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP. The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3' or 5' to the mismatch on an unmethylated strand. However, activation is sensitive to DNA topology. Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule. MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate. We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.

Adenosine Triphosphatases↗
PubMed1990

Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.

Nuclear extracts derived from HeLa and Drosophila melanogaster KC cell lines have been found to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision located 808 base pairs from the mismatch. Correction in both extract systems is strand specific, being highly biased to the incised DNA strand. Different mispairs within a homologous set of heteroduplexes were processed with different efficiencies (G.T greater than G.G approximately equal to A.C greater than C.C), and correction was accompanied by mismatch-dependent DNA synthesis localized to the region spanning the mispair and the strand break, thus demonstrating that mismatch recognition is associated with the repair reaction. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of high concentrations of ddTTP, indicating probable involvement of alpha and/or delta class DNA polymerase(s). These findings suggest that higher eukaryotic cells possess a general, strand-specific mismatch repair system analogous to the Escherichia coli mutHLS and the Streptococcus pneumoniae hexAB pathways, systems that contribute in a major way to the genetic stability of these bacterial species.

Animals↗
PubMed1989

Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease.

Gap repair in the presence of 2'-deoxycytosine 5'-O-(1-thiotriphosphate) has been utilized to mutagenize the amino-terminal one-half of the structural gene for EcoRI endonuclease. This approach has led to identification of over 200 mutants defective in endonuclease function. One mutant protein, which binds to the EcoRI sequence but displays greatly reduced cleavage activity, is the consequence of a Glu to Gly change at position 111. This protein has been purified to homogeneity and characterized in detail. Subunit interactions governing the tetramer to dimer transition of the mutant endonuclease are near normal as are parameters governing its interaction with specific and nonspecific DNA sequences. However, the rate constants for first and second strand cleavage steps are reduced by 60,000- and 30,000-fold, respectively, as a consequence of the Glu----Gly change. The defect in chemical cleavage steps can be partially overcome by elevating the pH of the reaction buffer from 7.6 to 8.5, conditions which enhance the rate of EcoRI* strand cleavage by wild type enzyme to a similar degree. We suggest that the Glu-111 mutation affects an interface between recognition and cleavage functions of the enzyme, an idea consistent with the suggestion that the cleavage center of the endonuclease is subject to activation upon specific recognition of the EcoRI sequence.

Base Sequence↗
PubMed1989

The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease.

King et al. (King, K., Benkovic, S. J., and Modrich, P. (1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition. We have substituted Gln or Asp for Glu-111 by oligonucleotide-directed mutagenesis. First and second strand cleavage rate constants are reduced by a factor of more than 10(4) by the Gln-111 substitution. However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI sites by wild type endonuclease to a similar degree. The specific affinity of Gln-111 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation. In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-111 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme. These results suggest that a negative charge on residue 111 is required for strand cleavage and are consistent with participation of Glu-111 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.

DNA, Bacterial↗
PubMed1989

DNA mismatch correction in a defined system.

DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites. The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates. This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located 1 kilobase from the mispair.

Base Sequence↗
PubMed1989

Methyl-directed DNA mismatch correction.

In 1964 Robin Holliday (1) proposed the correction of DNA base pair mismatches within recombination intermediates as the basis for gene conversion. The existence of the mismatch repair systems implied by this proposal is now well established. Activities that recognize and process base pairing errors within the DNA helix have been identified in bacteria, fungi, and mammalian cells. However, the functions and mechanisms of such systems are best understood in Escherichia coli, an organism that possesses at least three distinct mismatch correction pathways. These three systems are involved not only in the processing of recombination intermediates but also contribute in a major way to the genetic stability of the organism, a function anticipated for mismatch repair by Tiraby and Fox and by Wagner and Meselson. The significance of mismatch correction in the maintenance of low spontaneous mutability becomes apparent when one considers that seven E. coli mutator genes (dam, mutD, mutH, mutL, mutS, mutU, and mutY) have been implicated in mismatch repair. This minireview will summarize information on the most extensively studied E. coli system for mismatch correction, the methyl-directed pathway for processing of DNA biosynthetic errors and intermediates in genetic recombination. A discussion of other E. coli mismatch correction systems may be found in the recent literature and in several recent reviews. Mismatch repair pathways in other organisms and descriptions of the structural properties of mispaired bases may also be found in several of these reviews.

DNA Repair↗
PubMed1989

Isolation and characterization of the Escherichia coli mutL gene product.

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.

Bacterial Proteins↗
PubMed1989

Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs.

Mutations in the mutY gene of Escherichia coli confer hypermutability reflecting G.C to T.A transversion mutations and result in a deficiency in methyl-independent G-A to G.C mismatch correction. In the present work, the mutY product has been purified to near homogeneity by virtue of its ability to restore G-A to G.C mismatch correction to cell-free extracts of a mutS mutY strain. The 36-kDa protein renders the strand containing the mispaired adenine labile to base-catalyzed cleavage and sensitive to cleavage by several apurinic/apyrimidinic-site endonucleases, with the sites of strand scission by both agents corresponding to the location of the mismatch. These findings indicate that MutY is a DNA glycosylase that hydrolyzes the glycosyl bond linking the mis-paired adenine to deoxyribose. MutY, a 5'-apurinic/apyrimidinic-site endonuclease, DNA polymerase I, and DNA ligase are sufficient to reconstitute MutY-dependent G-A to G.C repair in vitro.

Adenine↗
PubMed1989

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.

DNA Repair↗
PubMed1988

Mismatch-containing oligonucleotide duplexes bound by the E. coli mutS-encoded protein.

The binding of the mutS gene product, a protein involved in at least two E. coli mismatch correction pathways, to a series of synthetic DNA duplexes containing mismatches or mismatch analogues of the purine/pyrimidine type was studied in order to establish whether a correlation exists between the recognition of these mispairs and the efficiency of their correction in vivo. Experiments using nitrocellulose filter binding or band-shift assays revealed that duplexes containing a G/T mismatch or its analogues I/T and DI/T were bound by the protein with affinities correlating to the efficiency of their repair in vivo. In contrast, the A/C mismatch, contained within the same sequence, was bound only poorly, despite being efficiently corrected in vivo. The analogues of the A/C mispair, uncorrected in vivo, were not detectably bound under the conditions of these assays.

Bacterial Proteins↗
PubMed1988

Mispair specificity of methyl-directed DNA mismatch correction in vitro.

To evaluate the substrate specificity of methyl-directed mismatch repair in Escherichia coli extracts, we have constructed a set of DNA heteroduplexes, each of which contains one of the eight possible single base pair mismatches and a single hemimethylated d(GATC) site. Although all eight mismatches were located at the same position within heteroduplex molecules and were embedded within the same sequence environment, they were not corrected with equal efficiencies in vitro. G-T was corrected most efficiently, with A-C, C-T, A-A, T-T, and G-G being repaired at rates 40-80% of that of the G-T mispair. Correction of each of these six mispairs occurred in a methyl-directed manner in a reaction requiring mutH, mutL, and mutS gene products. C-C and A-G mismatches showed different behavior. C-C was an extremely poor substrate for correction while repair of A-G was anomalous. Although A-G was corrected to A-T by the mutHLS-dependent, methyl-directed pathway, repair of A-G to C-G occurred largely by a pathway that is independent of the methylation state of the heteroduplex and which does not require mutH, mutL, or mutS gene products. Similar results were obtained with a second A-G mismatch in a different sequence environment suggesting that a novel pathway may exist for processing A-G mispairs to C-G base pairs. As judged by DNase I footprint analysis, MutS protein is capable of recognizing each of the eight possible base-base mismatches. Use of this method to estimate the apparent affinity of MutS protein for each of the mispairs revealed a rough correlation between MutS affinity and efficiency of correction by the methyl-directed pathway. However, the A-C mismatch was an exception in this respect indicating that interactions other than mismatch recognition may contribute to the efficiency of repair.

Base Composition↗
PubMed1988

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy.

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.

Binding Sites↗
PubMed1988

Methyl-directed DNA mismatch repair in Escherichia coli.

Some of the molecular aspects of methyl-directed mismatch repair in E. coli have been characterized. These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites. In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated. Analysis of mismatch correction in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process.

Adenine↗
PubMed1988

Escherichia coli mutY gene product is required for specific A-G----C.G mismatch correction.

A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of Escherichia coli [Su, S.-S., Lahue, R.S., Au, K.G. & Modrich, P. (1988) J. Biol. Chem. 263, 6829-6835; Lu, A.-L. & Chang, D.Y. (1988) Genetics 118, 593-600]. One is the mutHLS-dependent, methyl-directed pathway that recognizes a variety of mismatches and repairs the unmethylated strand of DNA heteroduplexes that are hemimethylated at d(GATC) sequences. The other pathway appears to be specific for A-G mispairs, yields C.G base pairs exclusively, and is independent of the presence of d(GATC) sites. Analyses of cell-free extracts prepared from E. coli mutY strains and isogenic parents have demonstrated that the mutY gene product is involved in the methyl-independent pathway, which converts A-G mispairs to C.G pairs. The specificity of this activity is consistent with the mutator phenotype associated with the mutY locus, which generates G.C----T.A transversions [Nghiem, Y., Cabrera, M., Cupples, C.G. & Miller, J.H. (1988) Proc. Natl. Acad. Sci. USA 85, 2709-2713]. We propose that the mutY product functions at a late stage of a pathway that excludes A-G mispairs during chromosome replication and that involves the function of the mutT gene product. This model suggests that the mutT function acts at an early stage of this pathway to exclude A-G mismatches where the adenine resides on the template DNA strand. A-G mispairs that persist after passage of the replication fork would contain guanine on the template strand and thus be processed to C.G base pairs by the mutY-dependent repair system.

Adenine↗
PubMed1987

Isolation and characterization of the Escherichia coli mutH gene product.

The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4639-4643) which is dependent on mutH function. The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini. Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand. Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations. d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions. These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.

Bacterial Proteins↗
PubMed1987

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family.

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83,303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch, W.J. and Feramisco, J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells.

Amino Acid Sequence↗
PubMed1987

Requirement for d(GATC) sequences in Escherichia coli mutHLS mismatch correction.

The involvement of d(GATC) sequences in Escherichia coli DNA mismatch correction was ascertained by analyzing in vitro repair efficiencies of a series of related, covalently closed circular DNA heteroduplexes that contained from zero to four d(GATC) sites. A heteroduplex with four d(GATC) sites was repaired with high efficiency by extracts of E. coli, whereas no significant correction occurred on a closely related molecule lacking such sequences. Heteroduplexes containing one or two d(GATC) sites were corrected at rates between 10% and 93% of that observed for the four-site molecule, but repair efficiency did not correlate in a simple way with the number of sites present. The methylation state at a single d(GATC) sequence was sufficient to direct strandedness of repair, and correction of heteroduplexes containing one or more d(GATC) sites required functional mutH, mutL, and mutS gene products. In addition, DNA repair synthesis dependent on mutH and mutS also required the presence of at least one d(GATC) site. Although mismatch correction was not observed on a covalently closed circular heteroduplex lacking a d(GATC) sequence, such molecules were subject to strand-specific repair if they contained a strand-specific single-strand break. However, this correction reaction did not require mutH, mutL, mutS, or uvrD gene products. Consequently, we have concluded that d(GATC) sequences are directly involved in mismatch correction mediated by the mutHLS system.

Base Sequence↗