Methods to Measure the Repair of Genes
Recently, there has been a lot of interest and many studies in the area of gene-specific repair. Demonstrations of DNA repair heterogeneity within the genome date back more than a decade. An early example of repair heterogeneity was the demonstration that repair incorporation in the first hours after UV damage was preferentially associated with the nuclear matrix (Mullenders et al., 1984). Other studies had shown DNA damage and repair heterogeneity in different subfractions of the cellular nucleus, within the genome, or in repetitive DNA sequences (reviewed in Bohr et al., 1987). A critical advance in this area of research was the development of a general technique to study the DNA repair process in individual genes (Bohr et al., 1985). This assay employed quantitative Southern blot analysis and it has at times been called the Bohr-Hanawalt technique. It is described below. The principal idea in this approach is to use a DNA repair enzyme to generate a strand break at the site of a lesion, and then to measure the frequency of DNA lesions within specific restriction fragments located within specific genes. Early experiments using this approach showed that genes could be repaired much more efficiently than the bulk of the DNA, and this led to the introduction of the concept of “preferential gene repair.” In the past years, numerous experiments have examined this technique and the use of strand-specific DNA probes in this same assay (Mellon et al., 1987) has led to new insight into the molecular link between DNA repair and transcription.
KeywordsChinese Hamster Ovary Cell Pyrimidine Dimer Strand Bias DHFR Gene Xeroderma Pigmentosum Group
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