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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B782-B787 (2003)
© 2003 The Gerontological Society of America

Effect of DNA Repair on Aging of Transgenic Drosophila melanogaster: I. mei-41 Locus

Sonia Symphorien and R. C. Woodruff

Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Aging appears to be increased by diminished DNA repair. To study this relationship between aging and DNA repair, we measured the life span of Drosophila melanogaster males in the absence of mei-41 excision repair and transgenic flies with 1 or 2 extra copies of the mei-41 wild-type gene. Life span was significantly reduced in the absence of repair and was significantly increased by an extra dose of excision repair. However, these changes in life span with alterations in DNA repair were not large.

THERE is extensive evidence that mutation is a factor in aging (1–5). Part of this evidence is from the observation that an increase in genetic damage in somatic cells reduces life span, whereas a decrease in genetic damage increases life span. For example, direct genetic damage induced by the movement of transposable DNA elements and by defects in DNA repair genes reduces life span (6–10), whereas a reduction in free radicals leads to an increase in life span (11,12). There are specialized protein complexes that sense DNA damage and signal other genes to arrest the cell cycle. This gives the cell time to repair the damage or, in some cases, to allow for apoptosis to occur (13). An example of such damage to DNA is double-strand breaks, which occur spontaneously in each cell (14). The vast majority of these breaks and other types of DNA damage are usually repaired, but defects in DNA repair can accelerate aging. For example, defects in DNA repair have been shown to decrease life span in Drosophila, mice, and humans (15–18).

The mei-41 repair gene of Drosophila melanogaster derives its name from the defect in meiotic recombination that results from a "weakly" mutated allele (19). The mei-41 protein produced by this gene is involved in meiotic recombination during the embryonic cell cycle and in DNA repair (20). Therefore, mutants of mei-41 have defects in meiotic recombination (21) and are mutagen sensitive (22–25). Furthermore, mei-41 has an essential role in embryonic development in a unique DNA replication/DNA damage checkpoint that is necessary for the transition from the maternally programmed early cell divisions to the zygotically controlled divisions after the midblastula stage (26). Mutations in the mei-41 gene of D. melanogaster were first identified on the basis of a defect in meiotic recombination (21) and, subsequently, by sensitivity to mutagens, including ionizing and ultraviolet radiation, methyl methanesulfonate, and hydroxyurea (22–25). Mutations in mei-41 also cause an increase in chromosome instability and breakage in mitotic cells and in the male germline (27–29).

The mei-41 and human ataxia telangiectasia proteins have similar structures and functions (20). Like mei-41 cells, ataxia telangiectasia cells are radiation sensitive, giving rise to increases in chromosome breakage (29,30). Moreover, the life span of cultured human cells with ataxia telangiectasia is reduced (31).

The role of mei-41 as a checkpoint protein extends beyond the sensing of DNA damage and includes the regulation of critical events in normal Drosophila development. Hawley and Friend (32) have also discussed similar roles for mei-41-like proteins in mammalian cells.

Do defects and extra doses of the wild-type mei-41 gene affect aging? It is the objective of this study to determine if flies with null mei-41 activity and with extra doses of the wild-type mei-41 gene have altered life spans. We observed that defects in mei-41 cause a clear reduction in life span and that an extra copy of the wild-type mei-41 gene increases life span.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Life span experiments were performed at 25°C using a dextrose medium of water, cornmeal, brewer's yeast, bacto-agar, dextrose, 10% lexgard, and 1.5% benzyl benzoate. Dextrose was substituted for sucrose in this life-span-storage medium to reduce the growth of a lactobacillus that produces mucus on the surface of standard cornmeal–molasses medium, which may trap flies (33). These vials were kept in the absence of light, except during aging counts and the transfer of flies to new vials.

Flies were etherized only on the first day of their lives. This allowed us to select males of each genotype, based on eye color. Fourteen flies were maintained per vial and were transferred to new vials with fresh food once a week.

Three runs were performed for each experiment, and surviving flies were counted on a daily basis. The results were incorporated into life tables, indicating the number of survivors in each vial per day. Any flies that escaped were eliminated from the experiment. Median and mean life spans (Tables 1 and 2) and daily survivorship curves (Figures 1 and 2) were determined for each male genotype of each run. The Kolmogorov-Smirnov test, as recommended by Mode and colleagues (34), and the combined probability test (35) were used to analyze our data.


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  		Table 1. Influence of the Wild-Type mei-41 DNA Repair Gene on the Lifespan of Drosophila melanogaster Males From Mating Scheme A.

 

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  		Table 2. Influence of the Wild-Type mei-41 DNA Repair Gene on the Life Span of Drosophila melanogaster Males From Mating Scheme B.

 


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  		Figure 1. Survival curves of Drosophila melanogaster males. Results from mating scheme A: effect of the absence of the wild-type mei-41 gene on life span compared with 1 dose and 2 doses of mei-41+. A, Run 1. B, Run 2. C, Run 3

 


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  		Figure 2. Survival curves for Drosophila melanogaster males. Results from mating scheme B: effect of the presence of extra doses of the wild-type mei-41 gene on life span compared with normal flies (1 dose of mei-41+). A, Run 1. B, Run 2. C, Run 3

 
Drosophila Stocks
The following mutants and transgenic strains were used in this experiment (36,37).

Mating Scheme A to Synthesize Males With 0, 1, and 2Copies of the mei-41 Wild-Type Gene
Males with no active mei-41 genes (w mei-41D3/Y; +/+) (white eyes), males with 1 active mei-41 gene (w mei-41D3/Y; P[w+ mei-41+]C1/+) (yellow eyes), and males with 2 active mei-41 genes (w mei-41D3/Y; P[w+ mei-41+]C1/P[w+ mei-41+]C1) (orange eyes) were collected from the following mating scheme (only F1 male progeny are shown).

Note that these 3 types of males have the same X, third, and fourth chromosomes. We tested these males for sensitivity to larval killing by methyl methanesulfonate. Previous experiments show that mei-41 mutant male larvae are sensitive to methyl methanesulfonate (22). The white-eyed males with no wild-type mei-41 gene were killed by this mutagen, whereas the yellow-eyed and orange-eyed siblings were more resistant to DNA damage.

Mating Scheme B to Synthesize Males with 1, 2, and 3 Copies of the mei-41 Wild-Type Gene
The following mating scheme was used to synthesize males with 1 copy (white eyes), 2 copies (yellow eyes), and 3 copies (reddish orange eyes) of the wild-type mei-41 gene. This mating scheme gives 3 different genotypes of male progeny: flies with the normal dose of mei-41+ (w mei-41+/Y; +/+), flies with 1 extra dose (w mei-41+/Y; P[w+ mei-41+]G1/+), and flies with 2 extra doses (w mei-41+/Y; P[w+ mei-41+]G1/P[w+ mei-41+]G1).

It is important to note that, in mating scheme A, 1 P element insert corresponds to 1 dose of mei-41 gene, and 2 P element inserts correspond to 2 doses of mei-41 gene. In mating scheme B, 1 P element insert corresponds to 2 doses of mei-41 gene, and 2 P element inserts correspond to 3 doses of mei-41 gene.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
The results from mating scheme A show that the absence of the wild-type mei-41 gene (w mei-41D3/Y; +/+) significantly reduces life span, compared with flies with 1 (w mei-41D3/Y; P[w+ mei-41+]C1/+ males) or 2 (w mei-41D3/Y; P[w+ mei-41+]C1/P[w+ mei-41+]C1 males) doses of mei-41+ in all 3 runs, except for one value in the second run where p =.051 (Table 1; Figure 1). However, flies with 2 doses of mei-41+ did not have a significant increase in life span compared with flies with 1 dose. In fact, flies with 1 dose of mei-41+ had a significantly longer life span than flies with 2 doses (Table 1; Figure 1; combined probability test for the 3 runs gives p <.01).

It seems that there is only a moderate decline in survivorship in the first few days of males lacking the mei-41 wild-type gene from mating scheme A runs 1 and 2 (see Figure 1A and C) as compared with males of mating scheme A run 2 (see Figure 1B). This may indicate that a deficiency in mei-41 is causing early death unrelated to aging in some crosses. However, if the data of mating scheme A is reanalyzed using only those males that lived at least 10 days, the life spans of the mei-41-deficient males are still significantly lower than the other 2-genotype males in 5 of the 6 comparisons in Table 1 (4 had p <.001, 1 had p <.01, and 1 had p =.08). Hence, even when one removes the males that died in the first 10 days, the mei-41-deficient males still have a significantly shorter life span than males with 1 or 2 doses of the mei-41 wild-type gene.

Data from mating scheme B show that an extra dose of mei-41+ increases life span. The life span in normal males with 1 dose of the wild-type mei-41 gene (w mei-41+/Y; +/+ males) was significantly lower compared with flies with 2 doses of mei-41+ (w mei-41+/Y; P[w+ mei-41+]G1/+ males) (Table 2; Figure 2), but was not significantly different from flies with 3 doses (w mei-41+/Y; P[w+ mei-41+]G1/P[w+ mei-41+]G1 males). In addition, flies with 3 doses of mei-41+ have a significantly reduced life span compared with flies with 2 doses (Table 2; Figure 2; combined probability test for the 3 runs gives p <.05).

Since eye color may influence life span in D. melanogaster, and the 3 types of males from mating schemes A and B have different eye colors, it is important to eliminate eye color as the cause of changes in life spans in this study. For example, in each of the 3 runs of mating scheme A, the yellow-eyed flies have the longest life span, whereas in each of the 3 runs of mating scheme B, the reddish-orange eyed flies have the longest life span. These results indicate that the presence or absence of DNA repair and not eye color is the cause of differences in life spans observed in this study.


   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The results from this study show that flies without the wild-type mei-41 gene have a reduction in life span, and that an extra dose of this DNA repair gene causes an increase in life span. In mating scheme A, the life span of males in the absence of the wild-type mei-41 DNA repair gene was significantly reduced compared with flies with 1 dose or 2 doses of the wild-type mei-41 gene. In mating scheme B, the life span of males with 1 extra dose of wild-type mei-41 was significantly increased compared with the normal wild-type males. Hence, an extra dose of wild-type mei-41 increases life span. However, in both cases, the presence of 2 extra doses of wild-type mei-41, corresponding to 2 P element inserts, decreases life span compared with flies with only 1 extra dose of mei-41+ (1 P element insert). Hence, in both mating schemes, 1 P element insert increases life span compared with flies without P elements, but the insertion of 2 P elements does not increase the life span as much as 1 P element insert. This could be due to a detrimental effect of extra mei-41 protein on development, or to the effect of the homozygous P element inserts (P/P) or to other homozygous second-chromosome mutations on life span, which is unrelated to the mei-41 effect. The 2 P inserts, or other mutations, may be altering normal gene activities, which in the homozygous P/P flies causes a reduction in life span.

The decrease in life span of D. melanogaster males defective in mei-41+ DNA repair observed in this study has been reported previously (6), giving support for the hypothesis that an increase in genetic damage in somatic cells, if not repaired, does reduce life span. There are 130 genes associated with DNA repair in humans (38). Would defects in any of these genes reduce life span? This interaction among repair genes and aging may be one of the reasons why numerous enzyme systems for the detection and repair of damaged DNA have evolved. It should also be noted that some of the deleterious mutations that are part of the evolutionary cause of aging might arise in repair genes.

The ability of species to repair DNA damage is directly related to life span (14). Repair of DNA damage in humans is more efficient than that in mice and other animals with shorter life spans (39–41). This suggests that DNA damage and repair may be a part of the aging process. It has also been shown that defects in DNA repair in humans that reduce life span can increase susceptibility to cancer (42,43). If DNA repair processes decline with age, this could be one reason why cancer is more common among older people (44). It is also of interest that some quantitative trait loci affecting life span in D. melanogaster map near DNA repair genes (45).

The results from this study clearly show that somatic genetic damage alters life span. Flies deficient in excision repair have a significant reduction in life span, and life span is extended by an additional dose of the wild-type mei-41 DNA repair gene. These results support the previous reported correlation between somatic mutation and aging. However, extra DNA repair does not always extent life span, as shown by Walter and colleagues (46) for transgenic mice with extra copies of the hMGMT DNA repair gene, although mice with defective repair do have a reduction in life span (17,18). Hence, it is important to test additional repair genes for their influence on the life span of transgenic Drosophila.



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  		Mating Scheme A

 


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  		Mating Scheme B

 

   Acknowledgments
 
The work was partially facilitated by National Aeronautics and Space Administration grant NAG2-1427. We thank Linda Treeger and Philippe Broutin for their technical help.

Address correspondence to R. C. Woodruff, Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403. E-mail: rwoodru{at}bgnet.bgsu.edu


   Footnotes
 
Decision Editor: James W. Curtsinger, PhD

Received February 3, 2003

Accepted April 18, 2003


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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