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Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and UVA irradiation.

ABSTRACT: Upon exposure to UVA light, psoralens can induce DNA interstrand cross-links (ICLs), which can block DNA replication and transcription. Among the psoralen derivatives, 8-methoxypsoralen (8-MOP) is conventionally applied for psoriasis therapy, and amotosalen S59 is used to inactivate bacterial and viral pathogens in blood components. In addition to the ICL formation, psoralens also readily form various monoadducts (MAs) with thymidine residues in DNA when exposed to UVA light, and the biological implications for these monoadducts remain unclear. Here, we reported a method that encompassed digestion with a single enzyme (nuclease P1) and LC-MS/MS, for the simultaneous quantification of ICL and MAs induced in human cells exposed with 8-MOP or S59 and UVA light. Our results showed that the yield of ICL induced by S59, which increased from 3.9 to 12.8 lesions/10(3) nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm(2), was approximately 100 fold more than that induced by 8-MOP. In addition, three and five products were identified as 8-MOP- and S59-MAs, respectively, and the yields of MAs were significantly lower than that for ICL. The yields of the three 8-MOP-MAs were 7.6-2.2, 1.9-9.9, and 7.2-51 per 10(6) nucleotides and those of the five S59-MAs were 215-19, 106-39, 25-21, 32-146, and 22-26 per 10(6) nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm(2). Although the yields of MAs induced by 8-MOP and S59 were lower than those of the respective ICLs under the same exposure conditions, the formation of appreciable amounts of MAs might account for some of the mutations induced by psoralens.

SUBMITTER: Lai C

PROVIDER: S-EPMC2626160 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and UVA irradiation.

Lai Congfang C Cao Huachuan H Hearst John E JE Corash Laurence L Luo Hai H Wang Yinsheng Y

Analytical chemistry 20081024 22

Upon exposure to UVA light, psoralens can induce DNA interstrand cross-links (ICLs), which can block DNA replication and transcription. Among the psoralen derivatives, 8-methoxypsoralen (8-MOP) is conventionally applied for psoriasis therapy, and amotosalen S59 is used to inactivate bacterial and viral pathogens in blood components. In addition to the ICL formation, psoralens also readily form various monoadducts (MAs) with thymidine residues in DNA when exposed to UVA light, and the biological ...[more]

PMID: 18947205

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Project description:Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.

Project description:Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli, we showed that colibactins directly generate DNA cross-links in cellulo Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.

Dataset Information

Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and UVA irradiation.

Publications

Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and UVA irradiation.

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