Nucleosomal Structure of Chromatin : Distribution and Excision of DNA Damage
The implications of the nucleosomal structure of chromatin for the distribution and repair of DNA lesions is of obvious interest. The “bulky” purine adducts formed in human cells by the highly reactive ultimate carcinogens N-acetoxy-2-acetylaminofluorene (AAAF), benzo(a)pyrene-diol-epoxide I (BPDE I) and benzo(a)pyrene-4,5-epoxide were all introduced in higher initial concentrations in nucleosomal linker- than in core-DNA. In contrast, comparable adduct concentrations in linker- and core-DNA were observed after prolonged exposure of actively metabolizing human lung cells to the procarcinogen benzo(a)pyrene. Since different distribution and repairability of structurally identical lesions may mean different biological potency extrapolation of results of in vitro mutagenesis and transformation using ultimate carcinogens to the in vivo situation where procarcinogens are metabolized in the target organ has to be done with caution.
No simple rule can be derived for the relative rates of adduct removal from core- and linker-DNA in human cells. While the BPDE I guanine adducts were removed with similar efficiency from both DNA fractions the AAAF adducts disappeared much more rapidly from linker DNA of confluent human fibroblasts. Under certain conditions large fractions of adducts persisted in the DNA over an extended period.
The observed non-random introduction of lesions into chromatin can be exploited to approach basic questions about the dynamic properties of chromatin in situ. Using AAAF and repair deficient confluent cultures of Xeroderma pigmentoswn fibroblasts we have obtained evidence which arques strongly against the existence of constitutive movement of nucleosomes. In a second series of experiments AAAF was used to probe the structure of Simian Virus 40 (SV 40) minichromosomes in situ in monkey kidney cells. It was found that the replication origin region of SV 40 DNA was more open to the attack by the agent than the rest of the genome. These results lend direct “in vivo” support to the electronmicroscopic observation that the origin region of isolated SV 40 minichromosomes was nucleosome depleted.
KeywordsMonkey Kidney Cell Human Lung Cell Repair Synthesis Staphylococcal Nuclease Normal Human Skin Fibroblast
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