Studies on the bonding specificity for mitomycin C-DNA monoalkylation processes

Li, Ven Shun; Kohn, Harold

doi:10.1021/ja00001a040

Cited by 87 publications

(78 citation statements)

References 1 publication

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, MRD appears to confer resistance by binding MC as a complex that prevents drug activation. The formation of MC-DNA adducts is highly efficient and selective for guanine nucleosides in the specific sequence 5Ј-CpG-3Ј (27,28). On the basis of an average GϩC content of 70%, there may be as many as 10 6 target sites within the genome of a typical streptomycete.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

et al. 1997

View full text Add to dashboard Cite

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an M r of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.Streptomyces species are gram-positive soil bacteria known for their ability to produce a wide range of biologically active metabolites. In addition, many resistance genes have been cloned from these bacteria. Mechanisms of cellular self-protection include drug inactivation, target site modification, reduction of intracellular concentration via efflux, and drug binding (8). The presence of multiple modes of self-protection toward a single antibiotic is well documented (9), often with one or more resistance determinants located adjacent to the corresponding biosynthetic genes.Mitomycin C (MC) was identified in 1956 as an antibiotic produced by Streptomyces lavendulae (18) and subsequently established as an important antitumor agent (19,21). MC functions as a prodrug and requires enzymatic or chemical reduction to become a highly reactive alkylating agent (19,40). The intracellular activation of MC is specified by endogenous flavoreductases (34) and proceeds by single electron reduction to the MC semiquinone radical. The relatively long-lived semiquinone species either rearranges to an alkylating intermediate (by further reduction) or transfers an electron to molecular oxygen to generate superoxide (32). Therefore, the ability of MC to inhibit bacterial and mammalian cell growth involves the combined action of DNA alkylation and the formation of reactive oxygen species. Other naturally occurring compounds within this class include bleomycin (37), enediynes (10), and the more recently discovered dihydrobenzoxazines (25,42).Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2, 3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23). In another example involving a DNA damaging agent, bleomycin self-resistance has been determined by drug modification (Bat) and binding (BLMA) proteins in Streptomyces verticillus (38). Beyond these examples, little is known about bacterial resistance to the growing cl...

show abstract

Section: Discussionmentioning

confidence: 99%

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

et al. 1997

View full text Add to dashboard Cite

show abstract

“…Formation of the monoadduct 2 is highly sequence dependent, with the sequence 5'-CG providing the best yield for thealkylation ofGua (Li & Kohn, 1991;Kumar et al, 1992). The base 3' to the Gua, on the other hand, has only a relatively modest effect.…”

Section: Atctctaaccatccc3$ -5'mentioning

confidence: 99%

Effect of site specifically located mitomycin C-DNA monoadducts on in vitro DNA synthesis by DNA polymerases

Basu¹,

Hanrahan²,

Malia³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

A series of site-specifically modified oligodeoxynucleotides were synthesized that contained either of the two known mitomycin C-DNA monoadducts. In vitro DNA synthesis was carried out on some of these templates using a modified bacteriophage T7 DNA polymerase (Sequenase), AMV reverse transcriptase, and two different varieties of Escherichia coli DNA polymerase I (Klenow fragment)--one that carries the normal 3'-->5' exonuclease activity and a mutant protein that lacks this enzymatic function. Regardless of the type of DNA polymerase being used, DNA synthesis was terminated nearly quantitatively at the nucleotide 3' to each of these two monoadduct sites, although primer extension to full length of the template was noted with the unmodified control template. Substitution of Mn2+ for Mg2+ at a high concentration of the deoxynucleotide triphosphates resulted in incorporation of nucleotides opposite the adduct in the incubations with Sequenase or the 3'-->5' exonuclease-free Klenow fragment; however, primer extension beyond the adduct site did not take place. These studies demonstrated that the mitomycin monoadducts are strong blocks of replication and are likely to be toxic lesions in vivo. Since previous molecular modeling studies and molecular mechanical calculations indicated that the mitomycin adduction does not induce severe distortions at the site of adduction, a lack of base-pairing ability of the modified base in the extended product is unlikely to be the reason for the inhibitory effect. Instead, energy-minimized structural models indicated that additional hydrogen-bonding interactions have been introduced by the mitomycin moiety, and perhaps this increased thermodynamic stabilization of a distorted structure of the replication fork, in turn, may block the replication bypass. Experimental evidence of increased thermodynamic stability was provided by thermal melting of a template/primer complex that presumably a polymerase encounters in a typical replication fork. Consistently higher Tm of the adducted "replication fork" was noted when compared to its unmodified counterpart.

show abstract

“…Alkylation and crosslinking are sequence-specific. Monoalkylation shows selectivity for guanine in the sequence 5'-CpG-3' (CpG) [33][34][35], while crosslinking is completely specific for CpG · GpC duplex sequences [36][37][38]. It has also been demonstrated that the efficiency and guanine selectivity of alkylation and crosslinking by mitomycin C is enhanced when the target guanines are base-paired with 5-methylcytosine [39][40][41][42].…”

Section: Mode Of Actionmentioning

confidence: 99%

Aziridine Natural Products – Discovery, Biological Activity and Biosynthesis

Lowden

2006

Aziridines and Epoxides in Organic Synthesis

View full text Add to dashboard Cite

Studies on the bonding specificity for mitomycin C-DNA monoalkylation processes

Cited by 87 publications

References 1 publication

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

Effect of site specifically located mitomycin C-DNA monoadducts on in vitro DNA synthesis by DNA polymerases

Aziridine Natural Products – Discovery, Biological Activity and Biosynthesis

Contact Info

Product

Resources

About