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PubMed1993

N-isopropyliodoacetamide in the reduction and alkylation of proteins: use in microsequence analysis.

A new reagent, N-isopropyliodoacetamide (NIPIA), for alkylation of sulfhydryl groups on proteins for microdigestion and microsequencing is described. The utility of this reagent in both of these procedures has been demonstrated. NIPIA was shown to be especially useful in microsequence analysis, where it yields high sensitivity in detection of Cys residues. This is because the phenylthiohydantoin (PTH) derivative of NIPIA-alkylated cysteine [PTH-Cys(NIPCAM)] appears as a sharp peak in a standard reverse-phase HPLC analysis of PTH amino acids, and elutes between PTH-Tyr and PTH-Pro where no other peaks are present. Thus the use of NIPIA circumvents various problems associated with HPLC analysis of PTH-Cys when other commonly used agents are employed for sulfhydryl alkylation, such as coeluting peaks or low signal levels. Procedures for the synthesis of NIPIA and other analogs, as well as PTH-Cys(NIPCAM), are also presented, and HPLC retention times for their corresponding PTH-Cys derivatives are compared.

Alkylation↗

PubMed1994

Quantitation of cysteine residues alkylated with 3-bromopropylamine by amino acid analysis.

A new versatile reagent, 3-bromopropylamine, for the quantitative analysis of cysteine residues in proteins and peptides is reported. When added to amino acid standards, the 3-bromopropylamine derivative of cysteine, S-3-aminopropylcysteine, elutes in a unique position on four different amino acid analysis systems without modification to their standard gradients. Optimized conditions for the complete alkylation of cysteines in proteins with 3-bromopropylamine are described. The S-3-aminopropylcysteine is stable to standard acid hydrolysis conditions used for amino acid analysis. Cysteine values are within 10% of the predicted value in the amino acid analysis of acid hydrolysates of known proteins based on quantitation with S-3-aminopropylcysteine. No evidence of alkylation of other amino acids by 3-bromopropylamine is apparent from the amino acid analysis of proteins alkylated under the optimal conditions. These results expand the application of 3-bromopropylamine to include quantitation of cysteine by amino acid analysis as well as the previously reported identification of cysteines by protein sequencing.

Alkylation↗

PubMed1997

Enhanced DNA alkylation activities of Hoechst 33258 analogues designed for bioreductive activation.

A series of analogues of Hoechst 33258, designed to be subject to bioreductive activation, were synthesized, and interactions between these compounds and pBR322 DNA were investigated. Compounds containing a quinone group reacted with DNA via two possible pathways in the presence of reductants NADH or NADPH: radical cleavage and DNA alkylation. The corresponding dimethoxy compounds, which are not subject to reduction, showed very weak DNA binding ability. The strength of alkylation reaction of the quinone derivatives is related to leaving group ability. Furthermore, the quinone compounds preferentially alkylate DNA at 5'-CG and TG sequences rather than at the AT sites preferred as binding sites of Hoechst 33258.

Alkylation↗

PubMed1998

Phase 2 trial of single agent ifosfamide/mesna in patients with platinum/paclitaxel refractory ovarian cancer who have not previously been treated with an alkylating agent.

Ifosfamide has been shown to possess modest activity in patients with platinum/cyclophosphamide refractory ovarian cancer. Current standard initial chemotherapy for ovarian cancer does not include an alkylating agent (paclitaxel substituting for cyclophosphamide). To evaluate the activity of ifosfamide in patients with refractory ovarian cancer who had not previously received an alkylating agent, 21 patients with platinum/paclitaxel refractory disease were treated with the drug as a single agent (1.8 g/m2/day x 3 days, with treatment repeated every 28 days). Treatment was reasonably well tolerated in most patients, although 1 individual was removed from study secondary to neurotoxicity. One patient exhibited an objective response of measurable disease, while a second individual had a major decrease in CA-125 levels (no measurable disease present) following therapy. An additional patient experienced disappearance of severe pelvic pain following treatment but failed to meet the criteria for a partial response. We conclude that ifosfamide has modest activity in platinum/paclitaxel refractory ovarian cancer. However, the level of effectiveness does not appear to be increased in individuals who are alkylating-agent naive, compared to previously reported experience in patients with prior exposure to this class of cytotoxic drugs (10-15% response rate).

Adult↗

PubMed1980

Formation and subsequent repair of alkylation lesions in tissues of rodents treated with nitrosamines.

There is evidence that the formation of O6-alkylguanine in DNA may be an important reaction in the induction of tumors by dimethylnitrosamine and related carcinogens. The removal of this product from DNA may protect against carcinogenesis. The removal process has been studied both in vivo after administration of labelled dimethylnitrosamine and in vitro by incubation of isolated enzyme preparations with alkylated DNA. It has been found that: 1. Activity removing O6-alkylguanine from DNA is present in a number of tissues, but is most active in liver. 2. High doses of dimethylnitrosamine (20 mg/kg) inhibit removal of O6-methylguanine from DNA in vivo completely in rat kidney and hamster liver and partially in rat liver. This loss of activity was also seen in extracts prepared from these tissues assayed in vitro. 3. Chronic exposure to low levels of dimethylnitrosamine led to an increase in the activity of the hepatic enzyme removing O6-methylguanine. 4. Hypophysectomy or thyroidectomy decreased the activity of the hepatic removal system for O6-methylguanine and treatment with growth hormone or thyroxin increased it. 5. Dimethyl- and diethylnitrosamine are rapidly absorbed from the upper part of the small intestine (but not from the stomach). The degree of alkylation observed in the liver was independent of whether the carcinogen was administered orally or by i.v. injection, but at doses of 0.1 mg/kg or less, the reaction with the kidney was much lower after oral administration. Therefore, at low oral exposures to the carcinogen, the liver, which has the greatest capacity to remove O6-methylguanine from its DNA receives a relatively greater proportion of the alkylation. These results are discussed in terms of the degree to which the DNA repair system removing O6-alkylguanine might contribute towards a threshold dose and that variations in this activity might be reflected in the magnitude of this threshold.

Alkylation↗

PubMed1988

Development of alkylating agent-resistant human tumor cell lines.

Survival curves and dose escalation studies of four representative human tumor cell lines exposed to the various alkylating agents are presented. With HN2, at a level of one log of cell kill there was a fivefold range in drug concentration required to achieve this degree of cell kill among the cell lines, from 4.5 microM for the SL6 lung adenocarcinoma to 22 microM for the SW2 small-cell lung carcinoma. Four logs of SCC-25 squamous carcinoma cells were killed by 100 microM CDDP; however, there was only about one log of SL6 cells killed by 500 microM CDDP. To kill one log of G3361 melanoma cells required 24 microM 4-HC and to kill one log of SCC-25 cells required 24 microM, approximately a 16-fold difference. The curves for cell kill by L-PAM appeared to be biphasic, with a break at about 100 microM. There was about a threefold range in drug concentration required to achieve one log of cell kill with L-PAM, from 60 microM in the SCC-25 cell line to 18 microM in the SW2 line. To kill one log of SCC-25 cells required 295 microM BCNU and to kill one log of SW2 cell required 120 microM, about a 2.5-fold difference. The range of maximally tolerated HN2 concentrations were from 1200 microM for the SL6 cell line, 48 times the initial concentration, to 300 microM for the SCC-25 line, 16 times the initial concentration. The G3361 line tolerated the highest level of CDDP, 1900 microM, 48 times the initial concentration. The SCC-25 line, on the other hand, tolerated only 600 microM, 30 times the initial concentration. The SL6 cell line maximally tolerated 36 times the initial concentration of 4-HC (1450 microM), whereas the SCC-25 cell line tolerated only 18 times the initial concentration (720 microM). The G3361 melanoma tolerated 1555 microM, 30 times the initial concentration of L-PAM, and the SCC-25 cell line tolerated 700 microM, 14 times the initial concentration. The SL6 cell line tolerated the highest concentration of BCNU, 4200 microM, 24 times the initial concentration. The SCC-25 cell line tolerated 1450 microM, 8 times the initial concentration. In all cases, the SCC-25 cell line was least able to tolerate exposure to increasing concentrations of alkylating agents. The SL6 and G3361 cell lines showed the greatest tolerance for increasing concentrations of alkylating agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗

PubMed1977

On the function of hormone receptors in the action of steroidal alkylating agents.

Steroidal alkylating agents are supposed to bind to steroid hormone receptors in target tissues. By this interaction the steroid portion might act as a carrier for the alkylating group. Three steroidal agents (phenesterin, estradiol mustard, dehydroepiandrosterone mustard) were tested for their capacity to combine with estrogen and androgen receptors in normal and malignant target tissues. For measuring steroid receptor complexes ager gel electrophoresis was used. All three compounds (added at a 10(3)-fold excess) revealed in vitro no competition to receptor sites (estrogen and androgen binding). After preincubation of intact tissue estrogen mustard was effective in inhibiting the binding of 3H-estradiol and dehydroepiandrosterone mustard reduced the uptake of 3H-5alpha-DHT. This may be due to liberating of estradiol and dehydroepiandrosterone, respectively, from the intact molecule. From these data it is unlikely that the cytostatic steroidal agents are more effective than other alkylating drugs.

Alkylating Agents↗

PubMed1985

Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. General considerations, the nitrosoureas and alkylating agents.

In vitro drug sensitivity of tumour biopsies is currently being determined using a variety of methods. For these chemosensitivity assays many drugs are required at short notice, and this in turn means that the drugs must generally be stored in solution. There are, however, a number of potential problems associated with dissolving and storing drugs for in vitro use, which include (a) drug adsorption; (b) effects of freezing; (c) drug stability under the normal conditions of dilution and setting up of an in vitro assay; and (d) insolubility of drugs in normal saline (NS) or phosphate-buffered saline (PBS). These problems are considered in general, and some recommendations for use of solutions of drugs in in vitro assays are suggested. The nitrosoureas and alkylating agents are also investigated in greater detail in this respect. The nitrosoureas are found to be very labile in PBS at pH 7, with 5% degradation (t0.95) occurring in 10-50 min at room temperature. These values are increased about 10-fold on refrigeration and about 5- to 10-fold on reduction of the pH of the medium to pH 4-5. At pH 7 and room temperature, t0.95 is observed in under 1 h with the alkylating agents nitrogen mustard, chlorambucil, melphalan, 2,5-diaziridinyl-3,6-bis(2-hydroxyethylamino)-1,4-benzoquinone (BZQ), dibromodulcitol, dibromomannitol, treosulphan, and procarbazine. Of the other alkylating agents, 4-hydroperoxycylophosphamide (sometimes used in vitro in place of cyclophosphamide), busulphan, dianhydrogalactitol, aziridinylbenzoquinone (AZQ), and dacarbazine have a t0.95 of between 2 and 24 h, while ifosfamide and pentamethylmelamine are both stable in aqueous solution for greater than 7 days. About half the drugs studied in detail have been stored frozen in solution for in vitro use, although very little is known about their stability under these conditions.

Alkylation↗

PubMed1988

An adaptive response to alkylating agents in Aspergillus nidulans.

A simple method is described for demonstrating adaptation to alkylation damage in Aspergillus nidulans. One wild type, two MNNG-sensitive, and one MNNG-resistant strain all showed improvement in colony growth when challenged with MNNG following appropriate inducing pretreatments. Other alkylating agents (MMS, EMS) could also adapt mycelium to later MNNG challenge, while 4NQO and UV could not. The inducible effect was not transmissible through conidia. A standard reversion assay based upon methG proved impractical for studying mutation frequencies during alkylation treatments owing to variations in MNNG resistance amongst revertants.

Adaptation, Physiological↗

PubMed1989

Effect of lonidamine on the cytotoxicity of four alkylating agents in vitro.

We examined the ability of lonidamine, which has been described as an inhibitor of cellular respiration and glycolysis, to enhance the cytotoxicity of alkylating agents to MCF-7 human breast-carcinoma cells. Lonidamine was increasingly cytotoxic to MCF-7 cells with increasing time of exposure. With a 12-h exposure, the IC50 for lonidamine was about 365 microM, and with a 24-h exposure it was about 170 microM. A drug concentration of 250 microM was chosen for use in the drug combination studies. Lonidamine appeared to have a dose-modifying effect on cisplatin (CDDP), producing increasingly supra-additive cell kill with increasing CDDP concentration. When simultaneously incubated with lonidamine for 1 h, 500 microM CDDP yielded a cell kill that was 2 log greater than additive cytotoxicity. Extending the exposure to lonidamine for 12 h after CDDP treatment led to a small, additional aliquot of cell kill of about 2.5-fold over the CDDP concentration range. Lonidamine also appeared to have a dose-modifying effect on melphalan cytotoxicity in the melphalan concentration range of 100-500 microM. Between concentrations of 10 and 100 microM melphalan, the drug combination survival after 1 h exposure fell within the envelope of additivity for the two agents. However, maintaining the presence of lonidamine for an additional 12 h increased the effect such that the combination was supra-additive over the entire concentration range of melphalan. Simultaneous exposure to 4-hydroperoxycyclophosphamide (4-HC) and lonidamine for 1 h resulted in greater than additive cell kill, and extending the lonidamine exposure period such that lonidamine was present during and 12 h after 4-HC treatment further increased this effect. Lonidamine had a moderate effect on the cytotoxicity of carmustine (BCNU) with a 1 h simultaneous exposure; however, this treatment combination reached greater than additive cytotoxicity only at the highest concentration of BCNU tested. Extending the lonidamine exposure time for an additional 12 h resulted in supra-additive cell kill over the BCNU concentration range. Therefore, when lonidamine was present during exposure to the alkylating agent and its presence was then extended for an additional 12 h, a synergistic cell kill was produced with all four alkylating agents tested.

Alkylating Agents↗

PubMed2000

DNA guanine-guanine crosslinking sequence specificity of isophosphoramide mustard, the alkylating metabolite of the clinical antitumor agent ifosfamide.

PURPOSE: The purpose of this investigation was to determine the base sequence specificity of isophosphoramide mustard (IPM), the alkylating metabolite of ifosfamide, by crosslinking of designed DNA oligomers in comparison with the clinical alkylating agents mechlorethamine (ME) (nitrogen mustard) and phosphoramide mustard (PM), the alkylating metabolite of cyclophosphamide. METHODS: IPM, as well as PM and ME were each reacted with three dodecameric duplexes, which were designed to detect interstrand crosslinking between guanines in 5'-GC-3' (I), 5'-GNC-3' (II) or 5'-GNNC-3' (III) sequences (N = A or T). RESULTS: All three agents preferentially react with 5'-GNC-3' target sequences. The 5'-GNNC-3' target sequence is less reactive by a factor of approximately 2.5- to 10-fold, while 5'-GC-3' is of even lower reactivity. CONCLUSION: These results indicate that all three agents show approximately equal preference for reaction with a 5'-GNC-3' target sequence in spite of the fact that IPM yields a 7-atom crosslink, while the other two agents yield 5-atom crosslinks.

Antineoplastic Agents, Alkylating↗

PubMed1998

Enhancement of irinotecan (CPT-11) activity against central nervous system tumor xenografts by alkylating agents.

Two major obstacles in the treatment of patients with central nervous system malignancies are drug resistance and host toxicity. The goal of combination chemotherapy is to achieve therapeutic effects that are more favorable than using a single drug alone, but without an increase in normal organ toxicity. The study reported here examined the combination of a topoisomerase I inhibitor, irinotecan (CPT-11), with three different alkylating agents: 1,3-bis(2-chloroethyl)-1-nitrosourea, busulfan, and cyclophosphamide. We evaluated the antitumor effects of these three combinations against a panel of human tumor xenografts derived from central nervous system malignancies, including adult high-grade gliomas (D-54 MG, D-245 MG) and a childhood ependymoma (D-612 EP). In replicate experiments, the alkylating agents were given on day 1 in doses varying from 10% to 75% of the dose lethal to 10% of the animals, and CPT-11 was given on days 1-5 and 8-12 in doses varying from 10% to 100% of the dose lethal to 10% of the animals. The antitumor effects of the various combinations ranged from less than additive (7.61 days below additive with 0.5 CPT-11 + 0.75 cyclophosphamide in D-54 MG) to statistically significant (P < 0.001) supraadditive effects (18.80 days above additive with 0.5 CPT-11 + 0.5 1,3-bis(2-chloroethyl)-1-nitrosourea in D-54 MG). These studies show that the combination of the topoisomerase inhibitor CPT-11 and alkylating agents may increase the antitumor effect in some cases well above additive with no increase in host toxicity (0/10 deaths in both experiments cited above) and should be considered for combination chemotherapy of central nervous system malignancies.

Adult↗

PubMed1998

Absence or decreased levels of the hMLH1 protein in human gastric carcinoma cell lines: implication of hMLH1 in alkylation tolerance.

Defective hMLH1 function has been increasingly associated with acquired cellular resistance to DNA alkylation damage in human colorectal and endometrial cancer cells. To investigate the relationship between the DNA alkylation tolerance and the hMLH1 status in human gastric carcinoma cells, we determined the cellular response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the mutational changes, and the expression of hMLH1 in 11 human gastric carcinoma cell lines. Of 11 cell lines, 4 (SNU-5, -16, -620, and -719) were sensitive, whereas 7 (SNU-1, -216, -484, -520, -601, -638, and -668) were resistant to the cytotoxic effect of MNNG. As determined by Western analysis, it was evident that all the MNNG-resistant cell lines except one (SNU-601) produced very low or undetectable levels of hMLH1 protein compared to the MNNG-sensitive cell lines. A homozygous non-sense mutation that resulted in truncated protein was found in one MNNG-resistant cell line (SNU-1). Therefore, to determine whether the sensitivity of cells to MNNG can be restored by exogenous expression of hMLH1 protein, wild-type hMLH1 cDNA was introduced into the MNNG-resistant cells (SNU-1). The cytotoxicity test showed that expression of exogenous wild-type hMLH1 protein caused an increase in sensitivity to the cytotoxic effect of MNNG. This restoration was confirmed by an increase in the cell population containing less than the G1 amount of DNA (cell death) in the wild-type hMLH1-transfected cells, as determined by flow cytometry analysis. Together our results suggest that (1) the absence or decreased level of wild-type hMLH1 protein may be a frequent event in the human gastric carcinoma cell lines, (2) such alterations in the hMLH1 protein are closely associated with the MNNG tolerance in the human gastric carcinoma cell lines, and (3) the hMLH1 protein participates not only in the repair of DNA mismatches but also in the mechanism of escape from the cytotoxic effects of DNA alkylation damage.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Lactandrate: a D-homo-aza-androsterone alkylator in the treatment of breast cancer.

The sensitivity of breast neoplasms to hormonal control provides the basis of novel investigational treatments with steroidal alkylators. An androsterone D-lactam steroidal ester, the 3beta-hydroxy-13alpha-amino-13,17-seco- 5alpha-androstan-17-oic-13,17-lactam, p-bis(2-chloroethyl)amino phenyl acetate (lactandrate) was synthesized and tested for antitumor activity against six human breast cancer cell lines in vitro and against two murine and one xenograft mammary tumors in vivo. A docking study on the binding interactions of lactandrate with the ligand-binding domain (LBD) of estrogen receptor-alpha (ERalpha) was inquired. In vitro testing of lactandrate cytostatic and cytotoxic activity was performed on T47D, MCF7, MDA-MB-231, BT-549, Hs578T, MDA-MB-435 breast adenocarcinoma human cell lines. In vivo testing was performed on two murine mammary tumors, the MXT tumor and CD8F1 adenocarcinoma, as well as on human mammary carcinoma MX-1 xenograft. Molecular modeling techniques were adopted to predict a possible location and interaction mode of the molecule into LBD. Lactandrate induced significantly high antitumor effect against all tested in vitro and in vivo models. The cell lines with positive ER expression found to be significantly more sensitive to lactandrate. Moreover, lactandrate found to be positioned inside the binding cavity with its steroidal moiety, whilst the alkylating moiety protrudes out of receptor's pocket. Lactandrate produced important anticancer activity on breast cancer in vitro and in vivo. Some correlation between ER and lactandrate effect was demonstrated. Docking studies provide the basis for the structure-based design of improved steroidal alkylating esters for the treatment of estrogen-related cancers.

Adenocarcinoma↗

PubMed1988

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation.

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.

Alkylation↗

PubMed1987

The effect of 2-(4-methyl-1-piperazinylmethyl) acrylophenone dihydrochloride on the alkylation of tubulin.

2-(4-Methyl-1-piperazinylmethyl) acrylophenone dihydrochloride (MPMAP) is a novel inhibitor of microtubule assembly in vitro and in vivo whose molecular mechanism of action has not been investigated (M. L. Mallevais, A. Delacourte, I. Lesieur, D. Lesieur, M. Cazin, C. Brunet, and M. Luyckx (1984) Biochimie 66, 477-482). We have examined the effect of MPMAP on the alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide) (EBI). MPMAP is a very potent inhibitor of tubulin alkylation by iodo[14C]acetamide. MPMAP gives half-maximal inhibition at a concentration of 15 microM. MPMAP also inhibits the alkylation of denatured tubulin and of aldolase, implying that it reacts strongly with sulfhydryl groups. MPMAP does not, however, interfere with formation by EBI of a crosslink between cysteines 239 and 354 in the beta subunit of tubulin, suggesting that these sulfhydryls are located in a cleft in the tubulin molecule.

Alkylation↗

PubMed1990

Selective expression of cytochrome P450 isozymes by 4-n-alkyl-methylenedioxybenzenes in rat lung cells.

Rat lung preparations were analyzed for cytochrome P450 expression as a function of pretreatment with 1 mmol/kg/day of various substituted n-alkyl-methylenedioxybenzenes (MDBs). Lung P450s were quantitated by Western blotting and visualized by immunocytochemical techniques using polyclonal antibodies directed against P450IA1/IA2 and P450IIB1/IIB2. Results demonstrated that n-hexyl and n-octyl-MDB (but not n-butyl-MDB) increased P450IA1 in lung microsomes (50 pmol/mg microsomal protein) compared to untreated controls and n-butyl-MDB-treated animals (6 pmol/mg). However, treatment with all of these MDBs concomitantly decreased P450IIB1 in rat lung microsomes, from 40 pmol/mg in untreated control lungs to below the detection limit of this method (less than 2 pmol/mg). In vitro metabolism assays with rat lung microsomes, utilizing 7,12-dimethylbenz[a]anthracene as substrate, confirmed the increase in P450IA1 by selected MDBs and the concomitant decrease of catalytically active P450IIB1. Immunocytochemistry of rat lungs with these same IgGs indicated selective cellular responses. n-Hexyl-MDB (but not n-butyl-MDB) increased P450IA1-like immunoreactivity, but the increase was specifically localized to Clara cells. Immunocytochemical results further showed that P450IIB1-like immunoreactivity disappeared entirely from alveolar type II cells, yet appeared unchanged in Clara cells relative to untreated controls. Increases in P450IA1 due to treatment with n-hexyl-MDB were associated with increases in P450IA1 mRNA as indicated by Northern blot experiments. In contrast, the decreased levels of P450IIB1, consequent to treatment with n-alkyl-MDBs, were not associated with altered levels of pulmonary P450IIB1 mRNA as determined by Northern blotting and solution hybridization assays. These results imply that n-alkyl-MDBs regulate pulmonary cytochromes P450 by both transcriptional and post-transcriptional mechanisms.

Alkylation↗

PubMed1979

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.

Alkylation↗