The effect of cytosine methylation on the structure and repair of carcinogenic DNA adducts.

摘要

The E. coli nucleotide excision repair complex, UvrABC, is able to repair a variety of DNA damage products. The repair efficiency of this complex appears to be influenced by the structure of the damage site. Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that requires cellular activation to a diol epoxide (BPDE) in order to react with DNA. It has previously been shown that BPDE preferentially modifies guanines in methylated 5'-CpG-3' hotspot sequences, and recent structural data provides evidence that a conformational change occurs in at least one of the four isomers of this adduct upon cytosine methylation. N-acetyl-2-aminofluorene (AAF) is a well-studied aromatic amine that also requires cellular activation in order to bind DNA. Both of these carcinogens are readily recognized by the UvrABC excinuclease. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show the effect cytosine methylation has on the rate of incision of carcinogenic DNA adducts by the UvrABC excinuclease. Incubation of the DNA with the UvrABC excinuclease in vitro reveals 157 differences in the efficiency of adduct incision depending on the methylation status of the cytosine 5' to the site of damage. Oligonucleotide incision of AAF was 2.7 times more efficient when the cytosine 5' of the adducted guanine was methylated as opposed to unmethylated, whereas the incision efficiency of only the (-)trans isomer of BPDE increased by approximately two times with the addition of cytosine methylation. All of the other BPDE isomers were less efficiently cleaved when methylated. Therefore, it appears that an adduct-dependent structural change occurs with the addition of cytosine methylation that alters DNA damage recognition by the UvrABC enzymes. Cytosine methylation also affects the solution structure of the (+) trans-BPDE isomer in duplex DNA. (+)trans-BPDE lies in the minor groove in an unmethylated sequence as has been described previously. When a 5-mC is located immediately 5' of the adducted guanine, the predominant adduct conformation lies in the minor groove; however, a secondary, unresolved conformation occurs as indicated by NMR line-broadening, specifically on the 5'-side of the lesion.