Biomedical subjects

J T Drummond

Publications and source records attributed to J T Drummond.

At least 19 recordsLinked to original sources

PubMed2001

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites.

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A.C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.

Base Pair Mismatch↗

PubMed2000

Human mismatch repair and G*T mismatch binding by hMutSalpha in vitro is inhibited by adriamycin, actinomycin D, and nogalamycin.

Loss of the human DNA mismatch repair pathway confers cross-resistance to structurally unrelated anticancer drugs. Examples include cisplatin, doxorubicin (adriamycin), and specific alkylating agents. We focused on defining the molecular events that link adriamycin to mismatch repair-dependent drug resistance because adriamycin, unlike drugs that covalently modify DNA, can interact reversibly with DNA. We found that adriamycin, nogalamycin, and actinomycin D comprise a class of drugs that reversibly inhibits human mismatch repair in vitro at low micromolar concentrations. The substrate DNA was not covalently modified by adriamycin treatment in a way that prevents repair, and the inhibition was independent of the number of intercalation sites separating the mismatch and the DNA nick used to direct repair, from 10 to 808 base pairs. Over the broad concentration range tested, there was no evidence for recognition of intercalated adriamycin by MutSalpha as if it were an insertion mismatch. Inhibition apparently results from the ability of the intercalated drug to prevent mismatch binding, shown using a defined mobility shift assay, which occurs at drug concentrations that inhibit repair. These data suggest that adriamycin interacts with the mismatch repair pathway through a mechanism distinct from the manner by which covalent DNA lesions are processed.

Alkylating Agents↗

PubMed2000

Catalytic chromatography.

Catalytic chromatography exploits both specific biological affinity and catalytic specificity to selectively purify enzymes. Two different applications are presented. Purification of EcoRI restriction endonuclease to apparent homogeneity was accomplished in a single step with significantly greater yield and purification than was obtained with affinity chromatography. An attempt to purify the multiple DNA polymerase activities of Escherichia coli was also developed. Five well-resolved peaks of DNA polymerase activity were fractionated. In this new chromatographic mode, the enzyme binds immobilized substrate coupled to a column in the absence of some required cofactor. When the missing cofactor is added, the enzyme converts substrate to product and selectively elutes from the column.

Catalysis↗

PubMed1999

Genomic amplification of the human DHFR/MSH3 locus remodels mismatch recognition and repair activities.

Mismatch recognition in human cells is mediated by two heterodimers, MutS alpha and MutS beta. MutS alpha appears to shoulder primary responsibility for mismatch correction during replication, based on its relative abundance and ability to recognize a broad spectrum of base-base and base-insertion mismatches. Because MutS alpha and MutS beta share a common component, MSH2, conditions that influence the expression or degradation of MSH3 or MSH6 can redistribute the profile of mismatch recognition and repair. MSH3 is linked by a shared promoter with DHFR, connecting two pathways with key roles in DNA metabolism. In a classic example of gene amplification, the DHFR (and MSH3) locus can become amplified to several hundred copies in the presence of methotrexate. Under these conditions, MutS beta forms at the expense of MutS alpha, and the mutation rate in these tumor cells rises more than 100-fold. The implications for cancer chemotherapy include a potential increase in mutability when tumors are treated with methotrexate, which could increase the frequency of subsequent mutations that influence the tumor's drug sensitivity or aggressiveness. Because processing certain types of DNA damage by the mismatch repair pathway has also been implicated in tumor sensitivity to agents such as cisplatin, changes in expression at the DHFR/MSH3 locus may have further relevance to the outcome of multi-drug treatment regimens.

Base Pair Mismatch↗

PubMed1998

Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha.

A human MSH2-human MSH3 (hMSH2.hMSH3) complex of approximately 1:1 stoichiometry (human MutSbeta (hMutSbeta)) has been demonstrated in several human tumor cell lines and purified to near homogeneity. In vitro, hMutSbeta supports the efficient repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches. Human MutSalpha (hMutSalpha), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single-nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSbeta. Thus, the repair specificities of hMutSalpha and hMutSbeta are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides. The hMutSalpha level in repair-proficient HeLa cells (1.5 microg/mg nuclear extract) is approximately 10 times that of hMutSbeta. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSalpha, the hMutSbeta level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSalpha and hMutSbeta, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10144-10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation.

Adenosine Triphosphatases↗

PubMed1998

Cobalamin-dependent methionine synthase and serine hydroxymethyltransferase: targets for chemotherapeutic intervention?

Chemotherapeutic drugs targeted at folate-dependent reactions have typically been directed at a limited number of target enzymes: dihydrofolate reductase, thymidylate synthase, and GAR and AICAR transformylase. This review discusses two other potential targets for chemotherapeutic inhibition: cobalamin-dependent methionine synthase and serine hydroxymethyltransferase. Brief reviews of the catalytic properties of these two enzymes are presented, and possible strategies for chemotherapeutic intervention are discussed.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair.

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

DNA Repair↗

PubMed1997

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

Animals↗

PubMed1997

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Cisplatin and adriamycin resistance are associated with MutLalpha and mismatch repair deficiency in an ovarian tumor cell line.

In contrast to parental A2780 ovarian tumor cells, extracts of one doxorubicin-resistant and two independent cis-diamminedichloroplatinum(II)-resistant derivatives are defective in strand-specific mismatch repair. The repair defect of the three hypermutable, drug-resistant cell lines is only evident when the strand break that directs the reaction is located 3' to the mismatch, and in each case repair is restored to extracts by addition of purified MutLalpha heterodimer. As judged by immunological assay, drug resistance is associated with the virtual absence of the MutLalpha MLH1 subunit and greatly reduced levels of the PMS2 subunit. These findings implicate a functional mismatch repair system in the cytotoxic effects of these antitumor drugs and may have ramifications for their clinical application.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent.

Adenosine Triphosphatases↗

PubMed1995

Characterization of nonradioactive assays for cobalamin-dependent and cobalamin-independent methionine synthase enzymes.

Methionine synthase enzymes catalyze methyl group transfer from 5-methyltetrahydrofolate to homocysteine to give methionine and tetrahydrofolate. Assays for this enzyme activity usually monitor transfer of a 14C-methyl group from the N5-position of methyltetrahydrofolate to homocysteine to produce 14C-methionine that must be purified by anion-exchange chromatography. Alternatively, tetrahydrofolate may be derivatized with a formylating agent under acidic conditions to produce methenyltetrahydrofolate. We report optimization of this reaction for assay of cobalamin-dependent methionine synthase to give an economical method for determining enzyme activity that does not require the use of radioactive compounds. By heating for 10 min in 1 N hydrochloric acid containing 12% formic acid, the enzymatic product tetrahydrofolate is converted into methenyltetrahydrofolate, which absorbs light at 350 nm, while residual substrate 5-methyltetrahydrofolate does not contribute to the absorbance at 350 nm. The assay allows the derivatized product to be characterized in situ with a minimal increase in volume upon acidification. The results of the spectrophotometric assay given here have been compared with the radioactive assay to confirm the validity of the derivatization under the assay conditions. We also report the extension of this assay method for use in activity measurements of cobalamin-independent methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1995

Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells.

A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutS alpha, also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HOT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutS alpha 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.

Amino Acid Sequence↗

PubMed1994

How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase.

The crystal structure of a 27-kilodalton methylcobalamin-containing fragment of methionine synthase from Escherichia coli was determined at 3.0 A resolution. This structure depicts cobalamin-protein interactions and reveals that the corrin macrocycle lies between a helical amino-terminal domain and an alpha/beta carboxyl-terminal domain that is a variant of the Rossmann fold. Methylcobalamin undergoes a conformational change on binding the protein; the dimethylbenzimidazole group, which is coordinated to the cobalt in the free cofactor, moves away from the corrin and is replaced by a histidine contributed by the protein. The sequence Asp-X-His-X-X-Gly, which contains this histidine ligand, is conserved in the adenosylcobalamin-dependent enzymes methylmalonyl-coenzyme A mutase and glutamate mutase, suggesting that displacement of the dimethylbenzimidazole will be a feature common to many cobalamin-binding proteins. Thus the cobalt ligand, His759, and the neighboring residues Asp757 and Ser810, may form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group in methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1994

Nitrous oxide degradation by cobalamin-dependent methionine synthase: characterization of the reactants and products in the inactivation reaction.

Cobalamin-dependent methionine synthase catalyzes the remethylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor. The enzyme is susceptible to inactivation by the anaesthetic gas, nitrous oxide, through either short-term exposure to high levels or chronic exposure to low levels of this agent. We have studied the chemical reaction wherein the bound cobalamin prosthetic group of the enzyme from Escherichia coli catalyzes the degradation of nitrous oxide. By poising the enzyme at low ambient potentials in an electrochemical cell, the concentration of enzyme in the highly reactive cob(I)alamin state can be controlled, and the observed rate of inactivation is directly dependent upon the amount of enzyme in this redox state. The inactivation consumes both nitrous oxide and electrons, while nitrogen gas is evolved. The inactivation process is also directly dependent upon the proton concentration, but the effect of pH was found to be on the enzyme, revealing a redox-linked base presumed to be on or near the cobalamin. The chemical reaction between enzyme-bound cob(I)alamin and nitrous oxide that leads to inactivation is therefore independent of pH. In a single turnover experiment, where enzyme-bound cob(I)alamin is generated from methylcobalamin by methyl transfer to the substrate homocysteine, the cob(I)alamin decays to cob(II)alamin, suggesting that the degradation of nitrous oxide involves one-electron reduction of nitrous oxide. We propose that the inactivation chemistry is likely to be a one-electron reduction of nitrous oxide, which leads to the formation of a highly reactive oxidant, such as hydroxyl radical, and subsequent enzyme damage.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1994

Nitrous oxide inactivation of cobalamin-dependent methionine synthase from Escherichia coli: characterization of the damage to the enzyme and prosthetic group.

Nitrous oxide, or laughing gas, is an anaesthetic agent that inactivates cobalamin-dependent methionine synthase. This enzyme uses the highly reactive, enzyme-bound cob(I)alamin oxidation state of the prosthetic group to effect methyl group transfer from 5-methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. The cob(I)alamin is capable of reductively degrading nitrous oxide, and here we characterize the modifications that occur to the Escherichia coli enzyme following electrochemical inactivation. Methionine synthase was inactivated on a milligram scale by equilibrating enzyme containing bound cob(II)alamin with a reduced electrochemical mediator to give the reactive cob(I)alamin state under an anaerobic atmosphere of nitrous oxide. The primary damage occurs to a 37.2-kDa domain that binds S-adenosylmethionine (AdoMet), and inactive enzyme can no longer be reductively methylated using AdoMet. The damage is oxidative, and it includes the covalent addition of the mediator, triquat, to the enzyme selectively at valine 1177, as well as the formation of a covalent cross-link between peptides containing the only two cysteines within this domain. Spectrally, the prosthetic group bound to inactive enzyme resembles cob(II)alamin, although some loss in absorbance is apparent. When the enzyme was reconstituted with [57Co]cobalamin and the inactivation repeated, the cobalamin was recovered unmodified in approximately 75% yield, but two products derived from the cobalamin were also observed. We interpret the finding of oxidatively modified products as strong evidence that reductive degradation of nitrous oxide releases a potent oxidant, presumably hydroxyl radical or its equivalent, that is capable of modifying sites proximal to the cobalamin.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1993

Electrospray mass spectrometric analysis of the domains of a large enzyme: observation of the occupied cobalamin-binding domain and redefinition of the carboxyl terminus of methionine synthase.

Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the methylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor. We have used electrospray mass spectrometry as a powerful tool for characterizing separable fragments obtained by proteolysis of this monomeric 136.1-kDa enzyme. A central 28.0-kDa domain, reported to bind the cobalamin, has been purified to homogeneity in 30% yield. We were able to detect the domain with bound cobalamin by electrospray mass spectrometry at neutral pH. Mass analysis of a 37.2-kDa carboxyl-terminal domain was grossly inconsistent with either of the two amino acid sequences from previously published DNA sequences. We then used electrospray mass spectrometry to analyze peptides generated by a lysyl endoproteolytic digest of a C-terminal fragment, and we have constructed a peptide map that accounts for > 95% of the peptide mass derived from this domain. The correct translational end of this protein (27 residues downstream from the previously predicted ultimate residue) has been established, and sequence conflicts within the two published DNA sequences have been resolved (GenBank Accession Number J04975). Resequencing the DNA near the carboxyl terminus ruled out a frameshifted reading of the DNA and suggested that a cytosine had twice been incorrectly inserted late in the reading frame. The strategies reported here for sequence confirmation, localization of coenzyme-binding regions, and identification of chemically modified peptides within a large protein are potentially applicable to the characterization of many other proteins.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1993

Assignment of enzymatic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli.

Cobalamin-dependent methionine synthase catalyzes methyl group transfer from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine, and the cobalamin prosthetic group serves as an intermediate methyl carrier. Enzyme possessing cobalamin in the cobalt(II) oxidation state is inactive, and this form is activated by one-electron reduction coupled to methylation by S-adenosylmethionine (AdoMet). The enzyme from Escherichia coli has been divided into separable fragments by limited proteolysis with trypsin, and the contribution of each of these fragments to substrate binding and catalysis has been evaluated. The 37.7-kDa carboxyl-terminal domain binds AdoMet, and this was demonstrated through covalent modification with radiolabeled AdoMet during ultraviolet irradiation. Following reductive activation with AdoMet, the enzyme was digested with trypsin and a 98.4-kDa amino-terminal fragment was isolated. It retained at least 70% of the activity of the intact enzyme and must therefore possess determinants sufficient for the binding of methyltetrahydrofolate and homocysteine, as well as residues required for catalysis. However, when the cobalamin was oxidized to the cob(II) alamin state, the 98.4-kDa fragment could not be reductively remethylated with AdoMet. A purified, 28-kDa domain within the 98.4-kDa fragment retained bound cobalamin and therefore must play a central role in catalysis, but the isolated 28-kDa domain retained no catalytic activity. Because AdoMet binds to a different domain of the protein than methyltetrahydrofolate and homocysteine, the enzyme probably uses conformational flexibility to allow the cobalamin access to the required methyl donor or acceptor at the appropriate time in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗