New methods for detection of low levels of DNA damage in human populations.

Abstract

The use of a postlabeling method to characterize and to detect infrequent base modifications in DNA is outlined. This method has the advantage that low levels of DNA modifications, approximately 1 modified base per 105 nucleotides, can be detected. Moreover, a broad spectrum of modification can be identified by using this methodology. The basis for the method involves transfer of a radioactive phosphate from the y position of ATP to the 5'-hydroxyl terminus of 3'-phosphoryl nucleotides that are derived from modified DNA by appropriate nuclease digestion.The second method involves use of a defined DNA sequence within human cells. The a sequence is used as a probe for DNA damage to specific nucleotides. The a DNA sequence is reiterated approximately 300,000 times in the human genome and exists in tandem arrays. It comprises approximately 1% of the entire genome. The reiterated sequence is sufficiently homogeneous to permit its use as a probe for a site specific in DNA damage.Examples of the application of both of these methodologies to DNA damage inflicted in human cells by chemicals and ultraviolet light are provided. DMS reacts with purines in DNA (2), the principal adducts formed being 7-methylguanine, 3-methyladenine and 1-methyladenine (3). Following DMS treatment, the DNA was isolated, and digested to nucleotides. The nucleotides were then postlabeled and were resolved by HPLC. DMS-methylated bases, in nucleotide form, were readily resolved from the four normal DNA bases and were detected along with two other minor products, 5-methylcytosine and uracil.