Recognition of specific DNA sequences by mitomycin C for alkylation

Kumar, Shiv; Lipman, Roselyn; Tomasz, Maria

doi:10.1021/bi00120a016

Cited by 86 publications

(124 citation statements)

References 28 publications

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“…Exposure to MMC results in alkylation and interstrand cross-links (20,26), which are mainly repaired by NER in E. coli (52). The M. smegmatis NER mutants differed in their sensitivities to MMC.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

et al. 2009

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In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.The success of Mycobacterium tuberculosis as a human pathogen lies to some extent in its ability to survive and replicate in macrophages (23). It is currently unknown how it manages to overcome the assault on its DNA by macrophagegenerated reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), which represent an otherwise very effective defense against intracellular pathogens (5,8,35). One possibility is that M. tuberculosis effectively detoxifies ROI and RNI. Alternatively, it may possess highly effective DNA repair machineries. In silico analyses of mycobacterial genomes including M. tuberculosis (6, 34), Mycobacterium leprae (7), Mycobacterium bovis (15), Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis (The Institute for Genome Research [http://www.tigr.org]) revealed the presence of genes encoding enzymes involved in DNA damage reversal, nucleotide excision repair (NER), base excision repair (BER), recombinational repair, nonhomologous end joining, and SOS repair. Surprisingly, mycobacteria are devoid of the mutLS-based postreplicative mismatch repair (MMR) system (34, 51), which is otherwise highly conserved throughout evolution and contributes to mutation avoidance by correcting replication errors resulting from nucleotide misincorporation and polymerase slippage (27,46). In addition, MMR inhibits recombination between nonidentical (homeologous) sequences and thus helps to control the fidelity of recombination (32, 41, 56).The finding that mycobacteria exhibit a general mutation rate comparable to that of MMR-proficient species suggests that they possess alternative or compensating strategies for mismatch recognition and MMR (51). It is conceivable, for example, that their replication and recombination fidelity is higher than in other prokaryotes. Alternatively, it is possible that the fidelity of these processes is controlled by one of the other DNA metabolic pathways, such as NER.Prokaryotic NER has been extensively studied in Escherichia coli. It is mediated by the UvrABC excinuclease enzyme complex and the helicase UvrD (43), a system cap...

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Section: Resultsmentioning

confidence: 99%

Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

et al. 2009

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“…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

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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...

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“…These results demonstrate conclusively a strong correlation between duplex formation and the extent of alkylation with FR66979, analogous to that observed with MC, suggesting that monoalkylation reactions are strongly favored in duplex DNA structures. 9 …”

Section: Duplex VII 5 H C C C T C a I C A A I A I A C I T A T I A I Tmentioning

confidence: 99%

“…7 More is known about the interstrand cross-linking reactions of 1±3 than about their abilities to either monoalkylate or form intrastrand cross-links in DNA. In contrast, MC has been shown to form both intrastrand cross-links (at d(GG)) 8 and monoadducts (at dG) 9 in DNA through N2 of dG. Preliminary evidence for the formation of monoadducts between FR66979 and DNA was ®rst obtained in a study of DNA interstrand cross-linking by FR66979.…”

Section: Introductionmentioning

confidence: 99%

Monoalkylation of DNA by reductively activated FR66979

Paz

Sigurdsson

Hopkins

2000

Bioorganic & Medicinal Chemistry

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AbstractÐThe antitumor antibiotic FR66979 has previously been shown to form interstrand cross-links in duplex DNA at the sequence [5 H -d(CG)] 2 , linking the exocyclic amino groups (N2) of deoxyguanosine (dG) residues. During the reaction of reductively activated FR66979 with DNA, products are formed which have electrophoretic mobility in denaturing polyacrylamide gels which is intermediate between that of unmodi®ed and interstrand cross-linked DNA. We show here that these products are monoadducts between FR66979 and DNA and provide strong evidence for the site of alkylation being N2 of dG. Moreover, the sequence selectivity of monoalkylation reactions between FR66979 and DNA containing either 5] 2 was observed to be ca. 5-fold less than for the related antitumor antibiotic mitomycin C (MC). The mechanistic implications of this result are discussed. Furthermore, it was demonstrated that contrary to a previous report, FR66979 requires DNA to be in duplex form for ecient monoadduct formation. #

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Recognition of specific DNA sequences by mitomycin C for alkylation

Cited by 86 publications

References 28 publications

Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

Characterization of the Mycobacterial NER System Reveals Novel Functions of theuvrD1Helicase

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

Monoalkylation of DNA by reductively activated FR66979

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