DNA repair in specific sequences in mammalian cells

Abstract

To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of a number of adducts in the non-transcribed, heterochromatic alpha DNA of monkey cells (by physically isolating the DNA) and also the removal of pyrimidine dimers in a number of genes in rodent and human cells (by an indirect assay using a dimer-specific endonuclease). In confluent cells, psoralen and aflatoxin B1 (AFB1) adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha is severely deficient. Adducts of N-acetoxyacetylaminofluorene (NA-AAF) are formed in slightly higher frequencies in alpha, and removal is slightly deficient. The removal of thymine glycols from alpha DNA in gamma-irradiated cells is proficient, as is repair synthesis elicited by exposure to methyl methane sulphonate, dimethyl sulphate, or 254 nm ultraviolet light (u.v.). Removal of AFB1 and NA-AAF adducts from alpha is enhanced by small doses of u.v. but not by X-rays or DMS. The quantum efficiency of conversion of psoralen monoadducts to crosslinks is much lower in alpha DNA. Taken together, these results suggest that the highly condensed chromatin structure of alpha hinders access of the repair system that acts on bulky adducts but not of systems for repair of specific base damage, u.v. damage may alter this chromatin structure directly or facilitate the action of some system that changes accessibility of chromatin to repair. The repair deficiencies are not observed in actively growing cells, in which chromatin structure may be less condensed due to DNA replication. We have also demonstrated preferential excision repair of pyrimidine dimers in active genes. Dimers are efficiently removed from the essential dihydrofolate reductase (DHFR) and hydroxymethylglutaryl CoA reductase genes in Chinese hamster ovary (CHO) cells and from the transcribed c-ab1 proto-oncogene in the mouse cells. Both cell types remove few dimers from their overall genomes or from sequences distal to the DHFR gene; dimers are also removed poorly from the non-transcribed mouse c-mos gene. In human cells, dimers are removed more rapidly from the DHFR gene than from the genome as a whole. However, repair is as deficient in this gene in XP-C cells as it is in the entire genome. These results suggest that resistance to DNA damage correlates better with repair of vital or active sequences than with overall repair levels and that mutagenic efficiency may vary according to the activity of the gene under study.