The reactivity of an unusual amidase may explain colibactin’s DNA cross-linking activity

ABSTRACTCertain commensal and pathogenic bacteria produce colibactin, a small molecule genotoxin that causes interstrand cross-links in host cell DNA. Though colibactin has been found to alkylate DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and β-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line. This transformation would incorporate two electrophilic cyclopropane warheads into the final natural product scaffold. Overall, this work provides a biosynthetic explanation for colibactin’s DNA crosslinking activity and paves the way for further study of its chemical structure.

The Phenanthridine-modified Tyrosine Dipeptide

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.

Formation, Repair, and Genotoxic Properties of Bulky DNA Adducts Formed from Tobacco-Specific Nitrosamines

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) andN′-nitrosonornicotine (NNN) are tobacco-specific nitrosamines present in tobacco products and smoke. Both compounds are carcinogenic in laboratory animals, generating tumors at sites comparable to those observed in smokers. These Group 1 human carcinogens are metabolized to reactive intermediates that alkylate DNA. This paper focuses on the DNA pyridyloxobutylation pathway which is common to both compounds. This DNA route generates 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine,O2-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytosine,O2-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxythymidine, andO6-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine as well as unstable adducts which dealkylate to release 4-hydroxy-1-{3-pyridyl)-1-butanone or depyriminidate/depurinate to generate abasic sites. There are multiple repair pathways responsible for protecting against the genotoxic effects of these adducts, including adduct reversal as well as base and nucleotide excision repair pathways. Data indicate that several DNA adducts contribute to the overall mutagenic properties of pyridyloxobutylating agents. Which adducts contribute to the carcinogenic properties of this pathway are likely to depend on the biochemistry of the target tissue.

O6-Methylguanine-DNA Methyltransferase in Tumors and Cells of the Oligodendrocyte Lineage

Background:Oligodendrogliomas respond to nitrosourea-based chemotherapy and are induced in rats following transplacental exposure to ethylnitrosourea, observations suggesting that neoplastic and normal cells of the oligodendrocyte lineage are “sensitive” to nitrosoureas. Nitrosoureas alkylate DNA at O6-guanine with repair mediated by O6-methyIguanine-DNA methyltransferase (MGMT). The cytotoxic and carcinogenic properties of the nitrosoureas appear related to MGMT activity.Methods:To explore why oligodendrogliomas respond to chemotherapy, we measured MGMT activity in five chemosensitive human oligodendrogliomas and in rat oligodendrocyte lineage cells. We also measured MGMT activity in rat astrocytes and compared the cytotoxic effects of carmustine (BCNU) on oligodendrocyte lineage cells and astrocytes.Results:Low levels of MGMT activity were found in five of five human oligodendrogliomas. Cultures of neonatal rat glia enriched for oligodendrocyte lineage cells also had low levels of MGMT activity, approximately one-third that found in astrocytes (p < 0.02), and oligodendrocyte lineage cells were more sensitive to BCNU than astrocytes.Conclusions:Low MGMT activity may contribute to the chemosensitivity of some human oligodendrogliomas and rat oligodendrocyte lineage cells also have low levels. If drug resistance mechanisms in tumors reflect the biochemical properties of their cells of origin, then normal glia may serve as a laboratory substitute for human glioma.

A study of the genotoxic potential of flavonoids using short-term bacterial assays.

Genotoxic activities of flavonoids (quercetin, rhamnetin, isorhamnetin, apigenin, luteolin) were investigated using two short-term bacterial assays. In the "repair test" in Salmonella typhimurium (strains TA1538 uvrB- and TA1978 uvrB+) the flavonoids studied did not introduce any damage into the DNA recognized by UvrABC nuclease (correndonuclease II). The results of the SOS-Chromotest in Escherichia coli K-12 strains PQ37 (tag+, alk+) and PQ243 (tagA, alkA) indicated that flavonoids only weakly induced the SOS system. The addition of a liver activation system (S9 mix) did not increase the mutagenic effect of the flavonoids tested. Two compounds: rhamnetin, isorhamnetin and their putative metabolites formed in the presence of the S9 mix did not alkylate DNA at N-3 of adenine.