7.6. EFFECT OF AGING AND DIETARY RESTRICTION ON GENE-SPECIFIC REPAIR
The major problem with the previous studies that have compared DNA repair in animals of different ages or on different diets is the assay, i.e. UDS, used to measure DNA repair. UDS is a relatively crude assay, which neither directly nor selectively measures the removal of a specific type of damage. Rather, UDS measures UV-induced non-replicative DNA synthesis. Thus, changes in the specific activity of the thymidine precursor pool or replicative DNA synthesis could affect the level of DNA synthesis measured as repair in the UDS assay. A second concern is the limited correlation between UV-induced UDS and the sensitivity of cells to UV-irradiation, which brings into question how accurately UDS reflects true DNA repair capacity of a cell. For example, no correlation was found between UV-induced UDS in mammalian cells and the viability of cells to UV-irradiation (42).
In addition, keratinocytes from old human subjects showed greater sensitivity to UV-irradiation even though no age-related difference was observed in UV-induced UDS (43). A third important limitation of the UDS assay for measuring DNA repair is that it measures overall genome repair.
Fig. 7.4. Removal of CPDs from the genome overall and from the DNA fragments containing the albumin and H-ras genes. Primary cultures of hepatocytes were irradiated at 10 J/m2 of UV-irradiation. Graph A shows the percentage of CPDs removed from the genome overall and the DNA fragments containing the albumin and H-ras. Graph B shows the percentage of CPDs removed from the transcribed and non-transcribed strands of the albumin fragment. The data are taken from Guo et al. (46).
This is a problem when measuring repair of DNA damage by the nucleotide excision repair (NER) pathway, e.g. UV-induced DNA damage, because NER is heterogeneous.
The first example of intragenomic heterogeneity in DNA repair was revealed when Zolan et al. (44) showed that the removal of DNA damage from transcriptionally silent α repeat DNA sequences in African green monkey kidney cells by the nucleotide excision repair pathway was much less than the overall genomic repair. Subsequently, Bohr et al. (45) developed a technique to measure repair of UV-induced DNA damage in specific genes, and using this technique, they showed for the first time that the removal of cyclobutane pyrimidine dimers (CPDs) was heterogeneous in specific genes.
Fig.7.5. Effect of aging and dietary restriction on removal of CPDs from the genome overall. Hepatocytes were isolated from 6- (•) and 24- (o) rats fed ad libitum, or 24- month-old rats fed a calorie-restricted diet (Δ). The percentage of CPDs removed from the genome overall was determined 12 and 24 hours after 10 J/m² of UV irradiation.
Differential repair of CPDs induced by UV-irradiation has been observed in mouse skin, human primary fibroblasts and a variety of mammalian cell lines, e.g. hamster, mouse, rat and human, as well as in E. coli cells (for review see 46). These studies have shown that UV-induced damage in transcriptionally active genes is repaired more efficiently than UV-induced damage in genes that are not expressed or silent regions of the genome. In addition, the efficient repair in transcribed genes is associated with the preferential repair of the transcribed strand. The rapid repair of the transcribed strand of active genes is defined as strand-specific repair, transcription-coupled repair or preferential repair (47).
Fig. 7.6. Effect of aging and dietary restriction on removal of CPDs from the albumin fragment. Hepatocytes were isolated from 6-(•) and 24- month- old (o) rats fed ad libitum, or 24- old month- old rats fed a calorie- restricted diet (Δ). The percentage of CPDs removed from either the double strand (Graph A) or the transcribed strand (Graph B) of the albumin fragment was determined 12 and 24 hours after 10 J/m² of UV irradiation.
The transcriptionally independent DNA repair is referred to as global repair or bulk genome repair (47). Using the technique developed by Bohr et al. (45), Richardson et al. measured repair of specific genes in primary cultures of non-dividing rat hepatocytes. In this study, DNA damage was induced by irradiating the primary cultures of hepatocytes with UV light, and the presence of CPDs was measured in a 21-kb BamHI fragment containing the albumin gene, a 14-kb BamHI fragment containing the H-ras gene and the genome overall (46). The removal of CPDs from the DNA fragment containing the albumin gene was significantly higher than that from the genome overall or the DNA fragment containing the H-ras gene as shown in Fig. 2A. Within 24 hours, approximately 67% of the CPDs was removed from the DNA fragment containing the albumin gene compared to less than 40% for the genome overall and the DNA fragment containing the H-ras gene.
The lower repair that we observed for the 14-kb fragment containing the H-ras gene is probably indicative of repair of the non-transcribed region of this fragment, since the H-ras gene makes up only 2.4-kb of the 14-kb fragment.
Primary cultures of hepatocytes removed CPDs from the transcribed strand of albumin fragment more efficiently than from the non-transcribed strand (Fig. 7.4B); however, no differences were observed in the repair of the two strands of the fragment containing the H-ras gene. These results demonstrate that primary cultures of non-dividing rat hepatocytes show differential repair of UV-induced DNA damage that is comparable to what has been reported for transformed, proliferating mammalian cell lines. More recently, it was compared the ability of hepatocytes isolated from young and old rats to repair UV-induced DNA damage in the albumin gene and the genome overall. The DNA repair in hepatocytes isolated from rats fed a calorie-restricted diet was also examined. The most important observation of this study was that the effect of aging and dietary restriction on NER is heterogeneous; it appears to differ between the transcriptionally active gene and the genome overall (Figs. 7.5 and 7.6).
For example, 24 hours after UV-irradiation, hepatocytes isolated from old rats showed a 40% decrease in the percentage of CPDs removed from the genome overall, and dietary restriction prevented this age-related decrease in the repair of the genome overall. However, age and dietary restriction did not affect the repair of the genome overall 12 hours after UV-irradiation (Fig. 7.5). In contrast, the rate (12 hours after UV-irradiation) of removal of CPDs from the albumin fragment was reduced approximately 40% in hepatocytes isolated from old rats compared to young rats, but the extent (24 hours after UV-irradiation) of repair was unaltered with age (Fig. 7.6).
The age-related decrease in repair of the albumin fragment was due to a decline in repair of the transcribed strand and was attenuated by dietary restriction (Fig. 7.6). However, age has very little effect on the repair of the non-transcribed strand of this fragment. The results from Richardson et al. studies also suggest that the age-related decrease in the repair of the genome overall is not due to an age-related deficiency in general repair proteins because the removal of CPDs from the non-transcribed strand of the albumin was not altered with age. It is possible that the age-related changes in the structure of the chromosome are responsible for the decrease in the repair of transcriptionally inactive genes/regions. For example, several studies suggest that chromatin becomes more condensed with age because of an increase in protein-DNA cross-links and internucleosome interaction (for review see 48).
In addition, Mozzhukhina et al. (49) showed that the chromatin template activity for α and β DNA polymerases declined with age. They suggested that this age-related change was due to an increased chromosomal condensation. Increased chromosomal condensation could also reduce the access of DNA repair proteins to the DNA and thereby decrease the repair of the lesion. Richardson et al. found, that hepatocytes isolated from old rats were defective in the repair of the genome overall 24 hours after UV-irradiation but not 12 hours after. A similar phenomenon was observed by Gaubatz & Tan (50). They injected mice of various ages with N-methyl-N-nitrosourea and measured the removal of induced 7-methylguanine from the genome of mouse kidney. Kidney tissue from young and old mice showed a similar rate of repair for the genome overall; however, the extent of repair was significantly decreased in kidney of old mice compared to young mice.
They suggested that the age-related decrease in the extent of repair was due to structural alterations in the chromatin of old mice. Are the age-related changes in DNA repair that has been observed physiologically important in aging? Data suggest that changes have been observed in DNA repair may play a role in aging because the age-related decrease in the repair of genome overall and the repair of the transcribed strand of the transcriptionally active gene are reversed by dietary restriction, which has been shown to retard aging and result in increased lifespans in rodents (29).
The potential importance of the age-related decrease in preferential repair in aging is illustrated by Cockayne's syndrome. This disease is associated with a loss of preferential repair of the transcribed strand of transcriptionally active genes (51). Interestingly, patients with Cockayne's syndrome display certain characteristics of normal aging, such as typical "old face", osteoporosis, atheroscleorosis and calcification of cerebral blood vessels (27). The decline in the repair of non-transcribed DNA also could be important in aging because most of DNA in the genome of mammalian cells is transcriptionally inactive. The age-related decrease in the ability of the cells to repair non-transcribed, silent regions of the DNA could play a role in the age-related accumulation of DNA damage and mutations, which have consistently been observed with increasing age (for review see 26).
The decrease in the repair of transcriptionally inactive genes/regions also could be an important factor in the age-related increase in cancer.