Research Grants Program Office

Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable. One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway. Examination of mutants in both BER and Nucleotide Excision Repair show that there is actually an overlap of repair by these two pathways for the removal of cytotoxic lesions in Escherichia coli. The enzymes removing damaged bases in the first step in the BER pathway are DNA glycosylases. The coding sequences for a number of methylpurine-DNA glycosylases (MPG proteins) were cloned, and a comparison of the amino-acid sequences shows that there are some similarities between these proteins, but nonetheless, compared to other DNA glycosylases, MPG proteins are more divergent. MPG proteins have been purified to homogeneity and used to identify their substrates ranging from methylating agents to deamination products to oxidatively damaged bases. The ligation-mediated polymerase chain reaction has been used to study the formation of alkylation damage, and its repair in mammalian cells. We have studied DNA damage in the PGK1 gene for a series of DNA alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine, Mechlorethamine, and Chlorambucil and shown that the damage observed in the PGK1 (phosphoglycerate kinase 1) gene depends on the alkylating agent used. This report reviews the literature on the MPG proteins, DNA glycosylases removing 3-methyladenine, and the use of these enzymes to detect DNA damage at the nucleotide level.