Dna Damage And Ageing Biology Essay

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Evolutionary theories suggest biological ageing is caused by accumulated stochastic damage in somatic cells. There is emerging evidence that suggests accumulated damage to DNA might be an important cause of biological ageing. Firstly, DNA lesions are observed to accumulate with age and there is a positive correlation between the lifespan of a species and its DNA repair rate. Moreover, and perhaps most convincingly, evidence comes from defects in DNA metabolism pathways which give rise to accelerated ageing phenotypes in various organisms. Despite this evidence, not all DNA repair defects accelerate ageing; insights from mutant mouse models into type of DNA damage important in ageing are emerging. Recent findings have also explored the role longevity-regulating genetic pathways in influencing the rate accumulation of DNA damage, it has been suggested that DNA damage drives functional decline with increasing age, but longevity mechanisms may be able to set how rapidly damage builds up.

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Future insights into the role of DNA damage in ageing might come from molecular epidemiological studies of populations for genes controlling genome stability. An interesting case of Brooke Greenberg, a 17 year old girl who has remained physically similar to toddler might shed further light on the role of genome stability in ageing.

A~n a~geing fa~ce Photo: GETTY CREA~TIVE

Contents Page

What is ageing? Evolutionary theories of ageing Page 3

DNA damage theory of ageing Page 5

DNA repair rate and maximum lifespan of a species Page 8

DNA lesions increase with age Page 9

DNA repair defects and accelerated ageing syndromes Page 10

Not all DNA repair defects accelerate ageing Page 12

New Insights Page 14

DNA damage & longevity pathways Page 17

Concluding remarks & future lines of research Page 18

What is ageing?

 

Initially, ageing appears to be a familiar concept, but upon detailed examination ageing means many things; it has social, behavioural, physiological and cellular aspects. Central to the biology of ageing is the premise that ageing is characterised by failure to maintain stability of the internal environment (homeostasis) under conditions of physiological stress, and this failure to maintain homeostasis is associated with a decrease in viability and an increase in vulnerability of the individual over time (Comfort, 1979).

In general, biological ageing can be defined as a series of time-dependent processes occurring in the adult organism that ultimately leads to its death. It represents a progressive decline in vitality and health with an increasing mortality risk common to nearly all metazoans. The ageing phenotype is said to be one of the most complex phenotypes, defined by generalised biological dysfunction.

Evolutionary theories of ageing

The cause and purpose of ageing can be examined at many levels, from a broad evolutionary perspective (ultimate causes), to the molecular level and biological mechanisms (proximate causes). One of the earliest explanations for the evolution of ageing was that senescence is programmed in order to limit population size or increase the turnover of generations. This was proposed to be adaptive for organisms living in changing environments, allowing for faster adaptation. The fundamental flaw of this early explanation of ageing was that there is little evidence that senescence is a significant cause of mortality in wild populations (Kirkwood, 2000). In contrast to protected populations, natural mortality is mostly due to extrinsic hazards such as predation, starvation and cold.

Three complementary theories attempt to provide an explanation for the ultimate cause of ageing. The mutation accumulation theory of ageing proposes that as a result of extrinsic mortality, there is a progressive weakening in the force of selection with increasing age, therefore mutations that affect health at older ages are not selected against. By an age when wild survivorship has declined to very low levels, the force of selection is too weak to oppose the accumulation of germ-line mutations with late acting deleterious effects (Kirkwood, 2000).

This explanation of ageing is similar to the evolutionary explanation of vision deterioration in cave animals: if some function cannot be used to provide reproductive advantage, it will not be supported by a selection pressure and therefore not maintained in future generations. The probability of an individual reproducing depends on its age. It is zero at birth and reaches a peak in young adults. Then it decreases due to the increased probability of death linked to various external (predators, illnesses, accidents) and internal (senescence) factors. In such conditions, deleterious mutations expressed at a young age are severely selected against due to their high negative impact on reproductive fitness (number of offspring produced). On the other hand, deleterious mutations expressed only later in life are relatively neutral to selection because their bearers have already transmitted their genes to the next generation (Gavrilov, 2002).

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A second theory - the antagonistic pleiotropy theory, takes this idea further by suggesting that differential effects of a gene (relative to age) can actually be caused by the same gene (pleiotropy). It proposes that genes beneficial at a younger age despite being deleterious at an older age will be favoured by selection and actively accumulated in populations. This theory is based on two assumptions. Firstly, it is assumed that a particular gene may have an effect not on one trait only but on several traits of an organism (pleiotropy). The second assumption is that these pleiotropic effects may affect individual fitness in opposite (antagonistic) ways.

This point can be illustrated by a hypothetical gene that increases the fixation of calcium in bones. Such a gene may have positive effects early in life because the risk of bone fracture and subsequent death is decreased, but such a gene may have negative effects later in life because of increased risk of osteoarthritis due to excessive calcification. In the wild, such a gene has no actual negative effect because most animals die long before its negative effects can be observed. There is then a trade-off between an actual positive effect at a young age and a potential negative one at old age; this negative effect may become effective only if animals live in protected environments such as zoos or laboratories (Gavrilov, 2002).

The antagonistic pleiotropy theory explains why reproduction may come with a cost for species longevity and may even induce death. Any mutations favouring more intensive reproduction (more offspring produced) will be propagated in future generations even if these mutations have some deleterious effects in later life. For example, mutations causing overproduction of sex hormones may increase the sex drive, libido, reproductive efforts, and success, and therefore they may be favoured by selection despite causing prostate cancer in males and ovarian cancer in females later in life. Thus, the idea of reproductive cost, or more generally of trade-offs, between different traits of the organism follows directly from antagonistic pleiotropy theory (Gavrilov, 2002).

The final evolutionary theory of the 'disposable soma' is a special case of the antagonistic pleiotropy theory. The theory proposes that somatic cells are only maintained to ensure continued reproductive success; following reproduction the soma is disposable. This theory postulates gene mutations with antagonistic pleiotropic effects. These hypothetical mutations save energy for reproduction by partially disabling molecular proofreading and other accuracy promoting devices in somatic cells. The authors of the disposable soma theory argued that "it may be selectively advantageous for higher organisms to adopt an energy saving strategy of reduced accuracy in somatic cells to accelerate development and reproduction, but the consequence will be eventual deterioration and death" (Kirkwood, 2000)

The theory argues that somatic maintenance need only be good enough to keep the organism in sound physiological condition for as long as it has a reasonable chance of survival in the wild. Since more than 90% of wild mice die in their first year, any investment of energy in mechanisms for survival beyond this age benefits at most 10% of the population. Nearly all of the mechanisms required for somatic maintenance and repair (DNA repair, antioxidant systems, etc.) require significant amounts of energy (ATP). Energy is scarce. The mouse will therefore benefit by investing any spare energy into thermogenesis or reproduction, rather than into better capacity for somatic maintenance and repair, even though this means that damage will eventually accumulate to cause ageing and ultimately death (Kirkwood, 2005).

DNA damage theory of ageing

One of the specific predictions made by the disposable soma theory is that ageing results from the accumulation of unrepaired cellular and molecular damage through evolved limitations in somatic maintenance and repair functions. The DNA damage theory proposes that it is damage to nuclear DNA that is of fundamental importance in causing ageing. The DNA damage theory of ageing proposes that ageing is a result of accumulated nuclear DNA damage in the cells of an organism. Nuclear DNA is subject to constant damage, mostly hydrolysis, oxidation and alkylation. In humans, DNA damage is very frequent (approximately 800 DNA lesions occur per hour in each cell) and several DNA repair processes have evolved to compensate (Lodish, 2004). These DNA repair processes and cell cycle checkpoint enzymes protect nuclear DNA by either repairing the damage or forcing cells into apoptosis or senescence.

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Some DNA damage remains in cells despite the action of the DNA repair processes and is accumulated over time. The accumulation of unrepaired DNA damage is more prevalent in certain types of cells, particularly in non-replicating or slowly-replicating cells, which cannot rely on DNA repair mechanisms associated with DNA replication such as those in the brain, skeletal and cardiac muscle. This accumulated DNA damage can contribute to ageing directly by increasing cell dysfunction or indirectly by increasing apoptosis and cellular senescence.

DNA is an extremely important biological molecule that must be kept intact, most importantly in the germline and proliferating cells for proper function. DNA is subject to continuous modification from both endogenous reactive chemicals, such as reactive oxygen species, and exogenous environmental factors, such as food agents, industrial genotoxins, and ultraviolet and ionizing radiation (Lindahl, 1993).

DNA is intrinsically unstable, experiencing spontaneous reactions. There are two major forms of spontaneous DNA damage (i) deamination of adenine, cytosine, and guanine, and (ii) depurination resulting from cleavage of the bond between the purine bases and deoxyribose, leaving an apurinic site in DNA. DNA is also damaged by products of a cell's metabolism essential to the viability of the cell. Metabolism generates reactive oxygen and nitrogen species, lipid peroxidation products, endogenous alkylating agents, estrogen and cholesterol metabolites, and reactive carbonyl species, all of which damage DNA. Reactive oxygen and nitrogen species alone generate several kinds of single-strand breaks and more than 70 oxidative base and sugar products in DNA (Hoeijmakers, 2009).

DNA is also damaged by exogenous physical and chemical agents. UV light induces the formation of pyrimidine dimers, in which two adjacent pyrimidines e.g., thymines are joined by a cyclobutane ring structure. Alkylation can be caused by exogenous chemicals in which there is an addition of a methyl or ethyl group to various positions on the DNA bases. Many carcinogens react with DNA bases, resulting in the addition of large bulky chemical groups to the DNA molecule.

Figure 1

(A~bdel, K)

DNA alteration can take two forms: mutation and DNA damage. A comparison illustrates the difference: the word STOP can be mutated to the word STEP by the substitution of another letter, whereas if the letter "O" is lost or altered, damage occurs, resulting in the non-word ST#P. Substitution of a Thymine for an Adenine would be a mutation, whereas methylation of a Guanine would be damage.

DNA~ da~ma~ge tends to interfere with gene expression by preventing tra~nscription of RNA~ from DNA~, wherea~s muta~tion usua~lly results in tra~nscription tha~t usua~lly produces proteins with diminished or a~ltered functiona~lity. Muta~tions tha~t a~re not letha~l to a~ cell a~re more likely to be perpetua~ted in dividing cells. DNA~ da~ma~ge ra~ther tha~n DNA~ muta~tion is thought to be the ca~use of a~geing.

Type of Damage

Events per cell per day

% of total daily damage

Single-strand break

120,000

50.9

N7-MethylGuanine

84,000

35.6

Depurination

24,000

10.2

O6-MethylGuanine

3,120

1.3

Oxidized DNA

2,880

1.2

Depyrimidation

1,320

0.5

Cytosine deamination

360

0.2

Double-strand breaks

9

0.01

Interstrand cross-links

8

0.01

Figure 2

(Benbest,P, 2009)

Sponta~neous DNA~ da~ma~ge is thought to be induced continua~lly through endogenous mecha~nisms a~nd from the environment, processed by the va~rious DNA~ replica~tion, repa~ir a~nd recombina~tion systems to ultima~tely genera~te muta~tions a~s a~ molecula~r end point.

Muta~tion a~ccumula~tion ma~y lea~d to cell dea~th, cell tra~nsforma~tion a~nd cell senescence, which in turn could underlie the va~rious symptoms of orga~nisma~l a~geing, including orga~n dysfunction, tissue degenera~tion a~nd a~ va~riety of pa~thologica~l lesions.

(J.Vijg, 2000)

Figure 3

The introduction of DNA damage usually has different immediate consequences from those occurring after the introduction of mutations. DNA damage is often recognized and removed by repair enzymes. Damage which is not immediately removed can interfere with the progress of DNA replication or transcription. Damage is not reproduced upon replication, but sometimes may persist in one strand through successive cycles of replication. Mutations can remain in the DNA indefinitely, affecting gene expression over succeeding generations.

DNA damage tends to interfere with gene expression by preventing transcription of RNA from DNA, whereas mutation usually results in transcription that produces proteins with diminished or altered functionality. Mutations that are not lethal to a cell are more likely to be perpetuated in dividing cells. Both damage and mutations can be removed, but by different mechanisms. Damage can be removed from individual organisms by repair enzymes; it can also be removed from a population of cells by causing death of the individual cells that have damaged DNA. Mutations can be removed from a population of cells or multi-cellular organisms through the consequences of their expression on the fitness of individual members of the population. Also, mutations may be removed infrequently by chance reverse mutation, or have their phenotypic effects moderated by suppressor mutations. There is also an important direct connection between DNA damage and mutation. During the processing of DNA damage by repair enzymes, mutations occasionally arise. Moreover, replication past unrepaired damage sites may result in mistakes leading to mutation (Bernstein, 1981).

DNA repair capacity correlates with lifespan

Studies comparing DNA repair capacity in different mammalian species have shown that repair capacity correlates with lifespan. This correlation provides indirect evidence that DNA repair capacity is an important factor in ageing. Hart and Setlow (1974) demonstrated that the ability of skin fibroblasts of seven mammalian species to carry out nucleotide excision repair after exposure to ultraviolet light correlated with the lifespan of the species.

Animal

Estimated lifespan

Shrew

1.5 years

Mouse

2 years

Rat

3.5 years

Golden Hamster

4 years

Cow

30 years

Elephant

70 years

Human

95 years

(Ha~rt & Setlow, 1974)

Figure 4

Figure 5

Figure 4 shows the ra~te of synthesis of DNA~ during non-S periods of the cell (mea~sure of the excision-repa~ir ra~te) for the different species.

Figure 6 shows the a~vera~ge lifespa~n of the ma~mma~lia~n species on a~ log sca~le is plotted a~ga~inst the extent of unscheduled DNA~ synthesis. The order of increa~se in unscheduled synthesis is the sa~me a~s tha~t of life-spa~n, suggesting Nucleotide excision repa~ir ca~pa~city is positively correla~ted with lifespa~n.

(Ha~rt & Setlow, 1974)

Figure 6

Further studies involving a wide range of mammalian species confirmed this relationship between lifespan and DNA repair capacity. Evidence came from studies of the enzyme Poly-ADP-ribose polymerase (PARP) which catalyses the post translational modification of proteins and plays a very important role in DNA repair acting as a regulator of base excision repair (BER) and also has a role in non-homologous end-joining (NHEJ) as well as in telomere maintenance.

Burkle et al. (1992) compared the PARP activities in mononuclear leukocytes from mammalian species of different maximal lifespans and showed a positive correlation between PARP activity and longevity of 13 mammalian species, with human cells showing about five times the PARP activity of rat cells (Grube & Burkle, 1992). A roughly linear correlation between maximum lifespan of mammals and DNA repair activity was found by a metaanalysis of five comparative studies which were re-analysed by standardising to an internal repair standard; promoting the suggestion that DNA repair rate is relevant to maximum lifespan (Cortopassi & Wang, 1996).

However, recent analysis by Promislow (1994) suggests that the positive correlation between mammalian lifespan and DNA repair rates may be an artefact of long-lived species being on average larger in body size. In mammals there is a strong correlation between adult body size and maximum lifespan. Promislow put forward a model that attempts to explain why there could be a correlation between body size and DNA repair rates. If we consider the following: individuals from two mammalian species of very different body size but similar lifespan, the assumption that DNA damage accumulation in a cell would eventually lead to fatal cancer, and a further assumption that both individuals want to maintain the same low probability of incurring that threshold level of DNA damage in a given period of time, the larger individual, having more cells, will need to maintain a higher rate of DNA repair to ensure that it has the same probability as the smaller individual in maintaining all its cells in a healthy state (Promislow, 1994). It is possible that large body size could have selected for high DNA repair rates and longer lifespan, giving rise to the observed correlations.

DNA alterations increase with age.

Further evidence for supporting the idea that accumulated DNA damage is responsible for ageing is the observation that DNA lesions and chromosomal abnormalities increase with age in healthy mammals. Martin et al. (1985) were able to demonstrate that there is a six-fold increase in the frequency of chromosomal aberrations in metaphase cells from old mice (40 months old) in comparison to young mice (8 months old). Curtis and Crowley provide further evidence for an increasing level of mutations during ageing. They looked at mouse liver parenchymal cell metaphase plates after partial hepatectomy and found considerably higher numbers of cells with abnormal chromosomes in old as compared to young animals (i.e., from about 10% of the cells in 4- to 5-month-old mice to 75% in mice older than 12 months). Later, such large structural changes in DNA, i.e., aneuploidy, translocations, dicentrics, were observed to increase with donor age in white blood cells of human individuals, i.e., from about 2%-4% of the cells being chromosomally aberrated in young individuals to about six times higher in the elderly. (Vijg J, 2000)

Sedelnikova et al. (2004) were able to show that -foci, which reveal DNA double-strand breaks accumulate in human cell cultures and ageing mice. These cryptogenic -foci remained after repair of radiation-induced -foci, suggesting that they may represent DNA lesions with unrepairable DSB. The study concluded that the accumulation of unrepairable double strand breaks may have a causal role in mammalian ageing.

DNA lesion accumulation may not reflect only the ongoing DNA damage induction but also a declining DNA-repair capacity over time. Once DNA damage has been generated, it is the role of cellular repair systems to prevent its accumulation. However, studies have shown that ageing is associated with a general reduction in DNA repair capacity. Weissman's team characterised several base excision repair enzymes in mammalian mitochondria. Base excision repair is the main pathway for the removal of small DNA base modification, such as alkylation and deamination. They compared the nuclear and mitochondrial base excision repair capacities between different mouse tissues by detecting and measuring the activities of three major DNA Glycosylases in the mouse brain. They observed a significant age-dependent decrease in uracil, 8-oxoG and 5-OH-C incision activities in the mitochondria of all brain regions, while variable patterns of changes were seen in nuclei (Weissman et al., 2007). Other studies have supported this observation of a decline in repair activities with age, for example Intano et al. (2003) which measured the base excision repair activity in mouse brain and live nuclear extracts. Des dpite these studies, the fundamental questions of causation remain; how can one distinguish if this decline in DNA repair and an increase in DNA damage is a cause of ageing rather than an effect of the ageing process?

DNA repair defects and accelerated ageing syndromes

The most compelling evidence in support of the DNA damage theory of ageing are a set of rare genetic disorders that appear to accelerate ageing. These disorders originate from mutations in the DNA damage repair and response genes.

Werner's syndrome (WS) is thought to be ca~used by muta~tions of RecQ type DNA~ helica~se. The syndrome is recognized a~s a~ top ra~nking 'segmenta~l' progeroid syndrome- the syndrome a~ppea~rs to a~ccelera~te a~geing in ma~ny tissues, but not a~ll. Pa~tients with WS show a~ wide va~riety of clinica~l a~nd biologica~l ma~rkers simila~r to norma~l a~geing a~t a~n ea~rly sta~ge of their life. WS pa~tients develop prema~ture greying, ca~ta~ra~cts, loss of subcuta~neous fa~t, skin a~trophy, osteoporosis, dia~betes, a~therosclerosis, a~nd ma~ligna~ncies, followed by dea~th a~t a~n a~vera~ge a~ge of 46. WS cells senesce prema~turely in culture a~fter just 20 divisions, compa~red to 60 divisions for norma~l huma~n fibrobla~sts (Fa~ra~gher RG, 1993).

Werner's syndrome

Ima~ge credit: Willia~m a~nd Wilkens Publishing inc.

Figure 7

WS originates in a recessive mutation in a gene WRN encoding the RecQ helicase. WRN is unique among its family in possessing an exonuclease activity (Huang et al, 1998) which indicates it may be involved in DNA repair. WRN plays a role in the maintenance of overall genomic stability, and may be involved in multiple DNA repair pathways (Bachrati and Hickson, 2003).

One important in vivo function of WRN is in homology-dependent recombination repair (HDR). HDR can be used to repair DNA damage while suppressing gene loss or rearrangement. WRN seems to have a role late in HDR when recombinant molecules are topologically disentangled for segregation to daughter cells. In mammalian cells, WRN seems to repair DNA-strand breaks that arise from replication arrest, and therefore functions to limit genetic instability and cell death.

WRN is proposed to function on DNA~ substra~tes tha~t a~re genera~ted during HDR repa~ir, either through sta~biliza~tion or repa~ir of replica~tion forks, or from telomere replica~tion or repa~ir. Successful resolution of these substra~tes suppresses genomic insta~bility a~nd ma~inta~ins telomere length a~nd structure to ensure high cell via~bility. In the a~bsence of WRN function, cells a~ccumula~te potentia~lly toxic DNA~ intermedia~tes or critica~lly short telomeres tha~t ca~n trigger genetic insta~bility, DNA~ da~ma~ge a~nd a~poptotic response pa~thwa~ys.

Figure 8

(Kudlow, BA~, 2007)

(Bria~n A~. Kudlow, 2007)

The model in figure 8 shows WS pathogenesis is driven by defective DNA metabolism that leads to genetic instability and mutagenesis. These consequences, together with mutation accumulation and cell loss, might drive the development of cell type or tissue-specific defects. Compromised tissue or organ structure and function then leads to seemingly divergent outcomes: senescence and apoptosis on one hand and mutation-dependent neoplastic proliferation on the other. It is unclear whether increased cellular senescence (Kipling D, 2004) or increased apoptosis (Pichierri P, 2001) is responsible for the progeroid symptoms.

One biogerontologist has challenged the concept of "accelerated ageing" on grounds that it is too easy to shorten lifespan with poisons and defective genes and too hard to validate in the absence of biomarkers of ageing that normal ageing has been accelerated (Miller RA, 2004) but microarray profiling of human fibroblast genes showed that the transcription alterations in WS were strikingly similar to those in normal ageing; 91% of the annotated genes displayed similar expression changes in WS and in normal ageing, 3% were unique to WS, and 6% were unique to normal ageing (Kyng KJ, 2003). This remarkable similarity between WS and normal ageing suggests that WS causes the acceleration of a normal ageing mechanism. This finding supports the use of WS as an ageing model and implies that the transcription alterations common to WS and normal ageing represent general events in the ageing process.

The protein whose muta~tion is responsible for the Hutchinson-Gilford syndrome is a~lso a~ nuclea~r protein. (Eriksson, M. et a~l. 2003). HGPS is the most severe of the progeroid syndromes. Individua~ls with HGPS ha~ve a~ mea~n lifespa~n of 13 yea~rs. HGPS pa~tients a~ppea~r norma~l a~t birth, but prema~turely develop severa~l fea~tures tha~t a~re a~ssocia~ted with a~geing, including a~lopecia~, a~therosclerosis, ra~pid loss of joint mobility, osteolysis a~nd elderly fa~cia~l phenotype (figure 9)  (Kudlow, 2007) but they ha~ve norma~l cognition a~nd immune function, a~nd no disposition to ca~ncer (Hennka~m RC, 2006)

(unc.edu)

Figure 9

The genetic basis of HGPS was uncovered in 2003, when it was found that most cases of the disease are associated with a single-nucleotide substitution that leads to aberrant splicing of LMNA, the gene that encodes the A-type nuclear lamins.(Eriksson, M. et al. 2003).

A-type lamins belong to the family of intermediate-filament proteins that, along with the B-type lamins, are the main constituents of the nuclear laminain. In addition to maintaining the integrity and shape of the nuclear envelope, lamin A/C has been implicated in the regulation of transcription and DNA replication (Kudlow, 2007).

Cells derived from patients with HGPS and HGPS mouse models display several indicators of an activated DNA-damage response, including enhanced phosphorylation of histone H2AX and markedly increased transcription of p53 target genes. A study which compared HGPS patient cells with the skin cells from young and elderly human subjects found similar defects in the HGPS and elderly cells. These defects included down-regulation of certain nuclear proteins and increased nuclear DNA damage (Scaffidi P, 2006).This data taken together strongly supports the conclusion that Lamina A is important in the physiology of ageing. DNA machinery is impaired in HGPS, suggesting that changes in the DNA are important in this disease and most likely in normal ageing.

(Kudlow, BA~, 2007)

Not all DNA repair defects accelerate ageing

Despite all the evidence supporting the role of DNA defects in ageing, there is also evidence to the contrary. Mice deficient in a DNA repair protein known as Pms2 have elevated levels of mutations in multiple tissues and yet did not appear to age faster than controls (Narayanan et al, 1997).

The Pms2 gene is one of several mammalian homologs of E coli MutL DNA mismatch repair gene. Mismatch repair gene homologs participate in various cellular functions, including transcription coupled repair and recombination; consequently a deficiency in one of these homologs may disrupt genome stability in multiple ways. Narayanan et al were able to construct transgenic mice that carried disruptions at Pms2 loci, in order to determine the effect of Pms2 inactivation on genome integrity in vivo. The transgenic mice defective in Pms2 showed 100-fold elevation in mutation frequency in all tissues examined compared to wild type mice. In accordance with DNA damage theory of ageing, it could be predicted that such profound genetic instability would cause accelerated ageing, but this was found not be the case in mice. The results showed that high rates of mutagenesis in multiple tissues are not necessarily associated with accelerated ageing. One possible explanation for this discrepancy is that DNA mismatch repair deficiency appears to be associated with small insertions/deletions in small mononucleotide repeat runs. It could be entirely possible that smaller mutations, i.e., point mutations and small deletions or insertions, are less important in creating a general spectrum of age-related degeneration than large rearrangement mutations. (J.Vijg, 2000) There was no consensus at the time on what type of DNA changes are crucial to ageing.

Moreover, in 1993 Cosgrove et al was able to demonstrate that X-irradiation of male germ line had no clear cut effect on the lifespan of subsequent offspring. Initially, this finding of no significant effect of a large dose of X-irradiation of mice on the lifespan of their progeny was a surprise. Subsequent X-ray studies by other investigators all support the finding showing no significant difference in length of life between offspring of control and irradiated fathers. It could be argued that X-rays could not accurately mimic the natural background of DNA damaging agents; more studies should be conducted on type of DNA damage rather the causative agent of such damage to ascertain what type of DNA damage is responsible for the accelerated ageing phenotype.

As figure 4 illustrates, the main DNA lesion caused by X-rays is single-strand breaks which are predominantly repaired base excision repair pathway and NER, it could be possible that single-strand breaks are tolerable for the cell and do not cause the profound genomic instability seen with other DNA lesions; this would explain why X-ray irradiated embryos do not show accelerated ageing phenotype.

If the DNA damage theory is correct it should be possible to delay ageing by enhancing DNA repair mechanism. In spite of several attempts the evidence is still lacking. (De Magalhaes, 2005a). There are no cases of delayed ageing in mammalian models that could be related to increased DNA repair. For example, the p48 gene provides UV-damaged DNA binding activity in cells and hence is very important in repairing damage derived from UV radiations. It was shown that rodents over expressing p48 had improved DNA repair mechanism and increased suppression of UV induced mutagenesis, but the rodents still did not live longer or age slower (Tang et al, 2000). Similar evidence was reported for another DNA repair protein MGMT involved in the removal of O6-methylguanine DNA lesion through direct reversal mechanism. Transgenic mice over expressing Human MGMT had significantly lower incidence of cancer but did not age any slower (Zhou et al, 2001).

If the progeroid syndromes do indeed represent a phenotype of accelerated ageing, then changes in DNA overtime in all probability play a crucial role in ageing. These diseases support the view that aging results from the accumulation of damage to cellular components most likely involved in DNA metabolism/repair caused by biochemical errors or deleterious agents. Despite this evidence, there remained uncertainty over what type of DNA damage is important in ageing phenotype and also the molecular mechanism by which DNA damage causes ageing.

New insights

New insights have emerged from genetic mouse models in identifying the mechanisms underlying progeroid syndromes. Some progeroid mouse mutants were found serendipitously while others were developed by mimicking human progeroid mutations in the mouse germ line. A detailed systematic analysis of these mice, compared with their littermate controls, revealed the premature appearance of various symptoms of ageing indistinguishable from the same phenotypes normally occurring much later in life (Garinis GA, 2008), suggesting that the mouse models constitute a valid ageing model.

These models helped establish that the consequences of different DNA lesions are determined by various parameters, such as the type of damage, frequency of lesions and their location in the genome. Some lesions were found to be primarily mutagenic, greatly promoting cancer, while others were mainly cytotoxic, triggering cell death or senescence which is thought to lead to degenerative changes such as those associated with ageing. The overall picture emerging from these mutant models is that genetic defects in DNA repair systems that mainly prevent mutagenesis are generally associated with a strong predisposition to specific types of cancer, with only minor symptoms of degenerative ageing. On the other hand, deficiencies in repair and surveillance pathways that mainly protect from the cytotoxic effects of DNA damage tend to be characterized by a decrease in the incidence of cancer and the premature appearance of some, but not all degenerative ageing phenotypes. Impairment of genome stability processes that combat both mutagenesis and cell death leads to susceptibility to both cancer and accelerated ageing, see figure 13 (Garinis GA, 2008).

Figure 10Disorders affecting genome maintenance fall into three categories: 1) conditions in which specific types of cancer are enhanced; 2) conditions in which many (but never all) aspects of ageing are accelerated, but cancer is reduced; 3) conditions in which both cancer and certain aspects of ageing are increased. The outcome seems to be governed by the genome maintenance system that is affected. See figures 10, 11, and 12.

Progeria~

Syndrome

Mutated genes

Affected Process

Cockayne Syndrome (CS)

CSA, CSB

Transcription coupled -NER

TC-NER; GG-NER

Trichothiodystrophy

XPB, XBD, TTDA

Partial GG/TC-NER

Cerebro-oculo-facio-skeletal syndrome

CSB, XBD, XPG

GG-NER; TC-NER

XPE

XPF/ERCC1

GG/TC-NER, ICL repair, HR

Rothmund-Thomson

RECQL4

Oxidative DNA damage repair

Dyskeratosis congenita

DKC1, TERC1

Telomerase maintenance

Hutchinson-Gilford progeria syndrome

LMNA

Nuclear lamina function

Atypical Werner syndrome

Restrictive dermopathy

LMNA, ZMPSTE24

Mandibuloacral dysplasia

Ca~ncer enha~nced

Figure 11

Syndrome

Mutated gene

Affected process

Breast cancer 1, early onset

BRCA1

DSB repair (HR)

Breast cancer 2, early onset

BRCA2

Li-Fraumeni

P53

Checkpoint control

Chk2

CHK2

G1 checkpoint control

Von Hippel-Lindau syndrome

VHL

Cell-cycle regulation

Hereditary non-polyposis colorectal cancer

Msh2, Mlh1

Mismatch repair

XP

XPC

GG-NER

Figure 12

Progeria~ + Ca~ncer

Syndrome

Mutated genes

Affected processes

Fanconi anaemia

FANC, BRCA2

DNA crosslink repair

Xeroderma Pigmentosum combined with CS

XPB, XPF, XPD, XPG

NER

Xeroderma Pigmentosum + DeSanctis-Cacchione syndrome

XPA, XPD

NER

Ataxia telangiectasia

ATM

DSB repair

Ataxia telangiectasia-like disorder.

MRE11

DSB repair

Nijmegen breakage syndrome

NBS1

DSB and Telomerase maintenance

Bloom syndrome

BLM

Mitotic recombination

Werner syndrome

WRN

Telomere maintenance, DNA recombination and repair

A~rrows pointing upwa~rd indica~te increa~ses in cell surviva~l or muta~genesis a~fter DNA~ da~ma~ge, a~nd a~rrows pointing downwa~rd indica~te decrea~ses; the grea~ter the number of a~rrows, the stronger the effect. NS denotes no significa~nt effect.

Figure 13

(Hoeijma~kers, 2009)

Double-strand breaks (DSBs) in DNA are highly cytotoxic form of damage. They are repaired through nonhomologous end-joining (NHEJ), which simply joins the ends of DNA strands or through homologous recombination (HR), which takes place after replication and uses the intact copy on the sister chromatid to properly align and seal the broken ends in an error-free manner. NHEJ promotes cellular survival in the presence of highly cytotoxic DSBs and may enhance mutagenesis (Hoeijmakers, 2009) while the HR promotes cellular survival, but without inducing mutations, figure 13.

Base-excision repair (BER) is involved with small DNA adducts some of which may be highly mutagenic (e.g., 7,8-dihydro-8-oxoguanine), and some cytotoxic. When these lesions block elongating RNA polymerase, transcription-coupled repair (TCR) removes the damage, allowing the vital transcription to resume. BER prevents mutagenesis and promotes cellular survival (Hoeijmakers, 2009).

Transcription-coupled nucleotide-excision repair is specific to transcription-blocking bulky adducts, which are eliminated throughout the entire genome by the global genome nucleotide-excision repair system. DNA damage that blocks the regular replication machinery involving DNA polymerase can be repaired by transcription couple repair which can affect survival but not mutagenesis or it can be bypassed by homologous recombination, which involves template switching and strand displacement, or bypassed by translesional synthesis (TLS), a specialized, relatively error-free but still somewhat mutagenic means of bypassing a specific subgroup of lesions.

Inspection of the nucleotide excision repair pathway gives us a very good illustration of why certain DNA repair defects lead to accelerated ageing, while other DNA repair defects show increased cancer formation. The nucleotide excision repair pathway is a multi-step 'detect, excise, and patch' repair system for a broad class of helix-distorting lesions such as UV-induced photoproducts and numerous bulky chemical adducts (Hoeijmakers, 2009). Such DNA damage is detected in two ways: 1) the global-genome NER (GG-NER) sub-pathway, which detects lesions with sufficient helix-opening properties anywhere in the genome, and 2) the transcription-coupled NER (TC-NER) sub-pathway, which is selective for lesions that stall the transcription elongation machinery (Garinis GA, 2008). Impairment of the global-genome nucleotide excision repair in humans causes Xeroderma Pigmentosum, characterized by an increase of more than 1000-fold in the susceptibility to sun induced skin cancer and an increased risk of internal tumours. This is explained by the fact that compromised global-genome nucleotide excision repair leads to accumulation of DNA lesions over the entire genome and with replication increases the risk of mutations and consequently cancer (Garinis GA, 2008). In contrast, impaired transcription-coupled nucleotide excision repair has little effect on mutagenesis: it repairs only occasional lesions that stall transcription, ignoring damage in the opposite, nontranscribed strand. Nonetheless, a defect in this repair mechanism underlies severe human progeroid disorders such as Cockayne's syndrome, which is characterized by early cessation of growth and development, severe and progressive neurodysfunction associated with demyelination, sensorineural hearing loss, cataracts, cachexia, and frailty. The average reported life span for patients with the disease is 12 years (Hoeijmakers, 2009).

Even though global genome repair is fully operational in people with Cockayne's syndrome, cells may die prematurely, suggesting that low levels of endogenous damage that block transcription and are not dispensed with quickly enough by other repair systems, and low levels of endogenous damage is sufficient to produce dramatic ageing phenotype.

The elimination of cells with low levels of damage protects the body from cancer at the expense of promoting ageing revealing a trade-off between these two outcomes of DNA damage. This shows us that there is a clear separation between the outcomes of cancer and accelerated ageing that result from DNA damage, depending on the type of repair process affected, and implies that mutations in isolation are not critical for the onset of ageing related diseases.

DNA damage & evolutionary conserved longevity pathways

The stochastic concept of ageing was experimentally challenged by the identification of loss of lifespan-extending single loss of function mutations. Mutations in the evolutionary conserved insulin/insulin-like growth factor signalling (IIS) pathway were found to extend the lifespan of adult worms (Guarente L, 2000). There is also compelling genetic evidence that indicates that the IGF-1 pathway is central to lifespan regulation and is evolutionarily conserved from primordial metazoans to mammals (Carter, 2002; Bartke A, 2001).

Suppression of this IGF axis in mammals is associated with delayed age related morbidity and longevity, profound metabolic changes including low serum glucose and insulin, enhanced antioxidant defences and stress resistance, and reduced frequency of somatic mutations (Liang H, 2003). It was noted that the onset of progeroid features in mouse models of human progeroid mutations was accompanied systematic suppression on the GH/IGF-1 pathway ( Niedernhofer, L, 2006). These changes also produced reduced serum glucose and insulin levels, a consistent upregulation of antioxidant defence and stress responses along with marked propensity to store glycogen and fat (Van de Ven, 2006). This was a paradoxical observation; short lifespan progeroid mice displaying the same profound metabolic changes as the longer lifespan caloric restricted mice mutants. It has been speculated that the progressive changes associated with the suppression of Insulin/IGF1 pathways and metabolism are adaptive responses aimed at minimizing further somatic damage, by shifting the energy equilibrium from growth and proliferation to preservation of somatic maintenance (Garinis, 2008) This adaptive survival response is likely to be driven by intrinsic genome stability, but it can also be triggered by other emergence situations such as caloric restriction, Although the author fails to suggest possible sequence of events leading from DNA damage to the activation of longevity assurance pathways. It has been suggested that the Damage drives functional decline with increasing age, but longevity mechanisms may be able to set how rapidly damage builds up.

Brooke a~ged 5

Brooke a~ged 10

Brooke a~ged 11

Brooke a~ged 16An interesting case of Brooke Greenberg, a child who is 16 years old, but whose size and development corresponds to that of an infant of 11 months might shed further light on the role of DNA in ageing.

(Wa~lker, 2008)

One the face of it, it appears Brooke Greenberg is frozen in time; however Walker et al (2008) found that different parts of her body and anatomy are maturing at different rates. For example her brain is hardly more mature than that of a newborn infant, yet her bones are around 10 years old, as determined by the maturity of the cells and structures. Despite being a teenager, she still has her baby teeth, with an estimated developmental age of about eight years. Genetic tests have shown that gene mutations associated with premature ageing in Werner's syndrome and HGPS are normal (Walker, 2008).  Further tests to determine mutations resulting in altered function of insulin/IGF-pathway might be able to shed more light in this case.

Concluding remarks and future lines of research

Numerous studies suggest that the DNA damage and the DNA damage response plays an important role in ageing, studies have begun to reveal the types of DNA damage might play a role in ageing, this however, has demonstrated the need for greater understanding of how increase senescence and apoptosis at the cellular level might bring about the ageing phenotype at an organismal level; more research is needed to elucidate the mechanisms by which senescent and apoptotic cells contribute to and bring about the ageing phenotype on an organismal level.

Additionally, evidence is beginning to indicate the possible link between DNA damage accumulation and evolutionary conserved longevity pathways in animals; more research into the mechanism of by which these conserved longevity pathways are able to alter the rate of accumulated DNA damage will surely give us great insight into the mechanism of the ageing process.

A line of evidence that would give great credibility to the DNA damage theory of ageing would be creating organisms with delayed ageing due to improved DNA repair capacity, to this end; efforts are underway to directly analyse human populations with varying ageing rates for polymorphic variation in genes controlling genome stability such as WRN and LMNA. In human populations there would expected to be allelic variation at the many loci harbouring genome stability genes to contribute to the observed variation in life span among individuals. It is conceivable that polymorphic variation at multiple loci could have given rise to genotypes conferring extreme longevity, for example, by optimization of genome stability and other cellular defence systems. Long-Lived individuals would show greater frequency of certain variant alleles indicating a potential functional advantage of those alleles. Protein bioinformatics methods could then help analyse the sequence and structural variation and provide insight into function.