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Structural analysis of a carcinogen-induced genomic rearrangement event.

ABSTRACT: We have explored the mechanism of genomic rearrangement in a hamster fibroblast cell culture system in which rearrangements are induced 5' to the endogenous thymidine kinase gene by chemical carcinogen treatment. The wild-type region around one rearrangement breakpoint was cloned and sequenced. With this sequence information, the carcinogen-induced rearrangement was cloned from the corresponding rearranged cell line by the inverse polymerase chain reaction. After the breakpoint fragment was sequenced, the wild-type rearrangement partner (RP15) was isolated by a second inverse polymerase chain reaction of unrearranged DNA. Comparison of the sequence of the rearrangement breakpoint with the wild-type RP15 and 5' thymidine kinase gene regions revealed short repeats directly at the breakpoint, as well as nearby A + T-rich regions in each rearrangement partner. Pulsed-field electrophoresis analysis demonstrated that this rearrangement is an interstitial deletion of 35 kilobases. Southern blot analysis of the RP15 region in unrearranged parental cells showed a demethylated CpG island and a complex of DNase I-hypersensitive sites adjacent to the breakpoint in the region deleted by the rearrangement. Therefore, these studies reveal interesting sequence and chromatin features near the rearrangement breakpoints and suggest a role for nuclear organization in the mechanism of carcinogen-induced genomic rearrangement.

SUBMITTER: Barr FG

PROVIDER: S-EPMC48361 | biostudies-other | 1992 Feb

REPOSITORIES: biostudies-other

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Structural analysis of a carcinogen-induced genomic rearrangement event.

Barr F G FG Davis R J RJ Eichenfield L L Emanuel B S BS

Proceedings of the National Academy of Sciences of the United States of America 19920201 3

We have explored the mechanism of genomic rearrangement in a hamster fibroblast cell culture system in which rearrangements are induced 5' to the endogenous thymidine kinase gene by chemical carcinogen treatment. The wild-type region around one rearrangement breakpoint was cloned and sequenced. With this sequence information, the carcinogen-induced rearrangement was cloned from the corresponding rearranged cell line by the inverse polymerase chain reaction. After the breakpoint fragment was sequ ...[more]

PMID: 1736310

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Project description:A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.

Dataset Information

Structural analysis of a carcinogen-induced genomic rearrangement event.

Publications

Structural analysis of a carcinogen-induced genomic rearrangement event.

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