2006 AFAR Research Grant: Caloric Restriction Represses DNA Damage to Replication Forks and Chromosomal Rearrangements

Please give a brief summary of your AFAR research project.
One of the aging theories, the disposable soma hypothesis, will be tested in the proposed research. It postulates that metabolic resources are optimally allocated between bodily maintenance/damage repair and physical growth/offspring reproduction. If energy, a form of metabolic resources, is not efficiently used for bodily maintenance/DNA damage repair functions, DNA damage remains unrepaired. Thus, with the repair functions progressively deteriorating, the accumulation of the damage may lead to aging. It is hypothesized that energy allocates to maintenance/repair functions during caloric restriction (CR); the allocation enhances repairing DNA damage. One type of DNA damage occurs during duplication of genetic materials– DNA replication. When DNA replication machinery stalls, DNA breaks and the broken fragments rejoin together in an order different from the original type (called rearrangements). The breaks and rearrangements are the events of genome instability. With special molecular techniques, the effects of CR will be investigated on the machinery stalling, rearrangements, and breakage in mutants that age faster than the normal cells. In the future, the location of rearrangements will be determined, and effects of cellular respiration function (mitochondrial integrity) will be studied on DNA replication.

In the current work, the antagonistic pleiotropy theory is being tested. It assumes that the genes, which affect multiple functions, may be beneficial and favored at young ages, but detrimental at old ages. The genes of this type are termed pleiotropic genes. In budding yeast, a gene called FOB1, encodes a replication blocking protein that stalls replication machinery and limits yeast lifespan. Thus, the FOB1 gene may be one of the pleiotropic genes. The roles of this gene and the protein in maintenance of lifespan and DNA replication are being investigated.

What problems are you addressing and what specific questions will your research seek to answer?
Aging is multifactorial; numerous theories have been postulated to explain the process. In the area of evolutionary genetics, the antagonistic pleiotropy theory postulates that there are special types of pleiotropic genes that have opposite effects on fitness at different ages, being advantageous in early life, but being harmful at later ages. However, evidence for existence of such genes is lacking. Our ongoing work is to address the problem.

The soma disposable theory suggests that energy allocation to bodily maintenance/repair of DNA damage or to energy storage/reproduction depends on the availability of food supply. When metabolic resources are reduced, lifespan can be extended by allocation of any spare sources to the maintenance instead of to reproduction or energy storage. Otherwise, investing resources into such processes as energy storage may reduce lifespan. Organisms may need a program for such metabolic allocation. This implies i) a connection of energy metabolism with soma repair of DNA damage and maintenance of genome stability, and ii) an aging-specific pattern of energy metabolism. Nonetheless, it is not completely understood whether energy allocation to maintenance/repair functions during CR enhances repairing damage to replication machinery. Our proposed investigation will contribute in this area.

What are the effects of CR on chromosome replication stalling? The disposable soma theory predicts that allocation of metabolic resources to maintenance of genome stability instead of energy storage enhances capacity of DNA damage repair during DNA replication. Indeed, energy metabolism is switched from energy storage to mitochondria-based respiration during CR. This CR-induced metabolic switch enhances stability of rDNA (a locus on chromosome XII of budding yeast) through Sir2 protein (a silent information regulator 2). The Sir2 protein contributes to the maintenance of rDNA stability and chromatin silencing, and connects energy metabolism to chromosome and lifespan maintenance. Another protein called Sgs1 is encoded by a yeast gene, which is homolog of human BLM and WRN. The patients who carry these mutates genes suffer pre-mature aging and cancer-susceptibility. We have found that chromosome replication stalls and breaks in the rDNA region in the yeast mutant Sgs1. This mutant, like the WRN human patients, is characterized by genome instability and premature aging. To address the research question, we propose that CR reduces replication stalling at the rDNA in the Sgs1 mutant via Sir2.

Does CR enhance repair of chromosome rearrangements and breaks? Eukaryotic genomes including humans' undergo rearrangements. These events may be exacerbated when replication machinery is slowed down moving in chromosomal regions containing repeated fragments, and when repair capacity is deteriorating in cells with impaired repair genes. Consistent with this, our preliminary results indicate that the size of yeast chromosome III is altered and breaks occur in rDNA in the Sgs1 mutant. We and others found that an increase in Sir2 levels represses replication stalling in the Sgs1 mutant, and CR enhances Sir2 activity. According to the results, we hypothesize that CR enhances repair of chromosomal breaks and rearrangements in the sgs1 mutant and the enhancement is mediated by Sir2.

What aspects of your project are most interesting from a scientific point of view?
An interesting aspect is that proposed investigation is aimed at elucidating molecular mechanisms connecting yeast energy, DNA metabolism and longevity. We have studied DNA replication stalling at the rDNA region and global chromosome rearrangements in various aging mutants. These mutants include long-lived cells with "harmful genes" mutated or pre-maturely aged yeast cells with repair and maintenance genes inactivated. We want to further investigate how energy metabolism and mitochondrial integrity affect DNA replication progression and genome stability in these long- and short-lived cells. The most interesting aspect is that results from the proposed investigation may be applicable to the study of human longevity, since human and yeast cells have similar molecular mechanisms for maintenance of genome stability.

What are the implications of your research for age-related diseases and disorders?
This proposed research, which is centered on metabolism and genome stability, will help us understand the molecular pathology of cancer-susceptibility/pre-mature aging diseases, such as Werner and Bloom syndromes. Moreover, the results are expected to provide insights into metabolic intervention such as low calorie diet as a means to maintain genome stability. The insightful finding will form molecular bases for development of anti-aging therapies for the age-related diseases, and in a broader sense, to enhance longevity and quality of senior life.

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