Site-specific DNA substrates for human excision repair: comparison between deoxyribose and base adducts

The genetic integrity of living organisms is maintained by a complex network of DNA repair pathways. Nucleotide excision repair (NER) is a versatile process that excises bulky base modifications from DNA. To study the substrate range of this system, we constructed bulky deoxyribose adducts that do not affect the chemistry of the corresponding bases. These novel adducts were incorporated into double-stranded DNA in a site-specific manner and the repair of the modified sites was investigated.

Using restriction enzymes as a probe for DNA modification, we confirmed that the resulting substrates contained the bulky deoxyribose adducts at the expected position. DNA containing these unique adducts did not stimulate DNA repair synthesis when mixed with an NER-competent human cell extract. Inefficient repair of deoxyribose adducts was confirmed by monitoring the release of single-stranded oligonucleotides during the excision reaction that precedes DNA repair synthesis. As a control, the same human cell extract was able to process a base adduct of comparable size.

Our results indicate that modification of DNA bases rather than disruption of the sugar-phosphate backbone is an important determinant for damage recognition by the human NER system. Specific positions in DNA may thus be modified without eliciting NER responses. This observation suggests new strategies for anticancer drug design to generate DNA modifications that are refractory to repair processes.