Cell, Vol. 120, 497512, February 25, 2005, Copyright 2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.01.

028

DNA Repair, Genome Stability, and Aging

David B. Lombard,1,2 Katrin F. Chua,1 Raul Mostoslavsky,1 Sonia Franco,1 Monica Gostissa,1 and Frederick W. Alt1,* 1 Howard Hughes Medical Institute The Childrens Hospital Department of Genetics Harvard Medical School and The CBR Institute for Biomedical Research Boston, Massachusetts 02115 2 Department of Pathology Brigham and Womens Hospital Boston, Massachusetts 02115

Review

ies per cell. Many mutants with phenotypes that resemble premature aging possess defects in nuclear DNA repair. Therefore, we focus on the role of nuclear DNA damage in mammalian aging, emphasizing recent work in mouse models. DNA Damage, Reactive Oxygen Species, and Aging A large body of evidence argues that DNA damage and mutations accumulate with age in mammals (Vijg, 2000). Cells harboring mutations at defined loci have been shown to increase with age in humans and mice. Cytogenetically visible lesions such as translocations, insertions, dicentrics, and acentric fragments also accumulate in aging mammalian cells. Mice with integrated reporter arrays have allowed estimates of the age-related occurrence of both point mutations and larger genomic rearrangements. Such analyses have revealed considerable variation in mutation spectra between different tissues. These differences likely reflect functional characteristics of those tissues, such as mitotic rate, transcriptional activity, metabolism, and the action of specific DNA repair systems. Reactive Oxygen Species: An Important Source of Age-Related DNA Damage There are many sources of DNA damage. In addition to external sources, such as ionizing radiation and genotoxic drugs, there are also cell-intrinsic sources, such as replication errors, spontaneous chemical changes to the DNA, programmed double-strand breaks (DSBs) (in lymphocyte development), and DNA damaging agents that are normally present in cells. The latter category includes reactive oxygen species (ROS), such as superoxide anion, hydroxyl radical, hydrogen peroxide, nitric oxide, and others. Major sources of cellular ROS production are the mitochondria, peroxisomes, cytochrome p450 enzymes, and the antimicrobial oxidative burst of phagocytic cells. ROS can cause lipid peroxidation, protein damage, and several types of DNA lesions: single- and double-strand breaks, adducts, and crosslinks. The situation in which ROS exceed cellular antioxidant defenses is termed oxidative stress. As normal byproducts of metabolism, ROS are a potential source of chronic, persistent DNA damage in all cells and may contribute to aging (Sohal and Weindruch, 1996). The ROS theory of aging is discussed in depth in this issue of Cell by Balaban et al. (2005). In brief, longer-lived species generally show higher cellular oxidative stress resistance and lower levels of mitochondrial ROS production than shorter-lived species. Caloric restriction, an intervention that extends life span in many organisms, likely decreases ROS production (Barja, 2004). In model organisms, many mutations that promote longevity concomitantly increase oxidative stress resistance (Finkel and Holbrook, 2000). In addition, levels of 8-oxoguanine (oxo8dG), a major product of oxidative damage to DNA, accumulate with age (Hamilton et al., 2001). The potential importance of oxidative damage to DNA in age-related dysfunction is highlighted by a re-

Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation. Introduction Although aging is nearly universally conserved among eukaryotic organisms, the molecular mechanisms underlying aging are only beginning to be elucidated. A useful conceptual framework for considering the problem of aging is the Disposable Soma model (Kirkwood and Holliday, 1979). This model proposes that organisms only invest enough energy into maintenance of the soma to survive long enough to reproduce. Aging occurs at least in part as a consequence of this imperfect maintenance, rather than as a genetically programmed process. Although aging may involve damage to various cellular constituents, the imperfect maintenance of nuclear DNA likely represents a critical contributor to aging. Unless precisely repaired, nuclear DNA damage can lead to mutation and/or other deleterious cellular and organismal consequences. Damage to both nuclear DNA, which encodes the vast majority of cellular RNA and proteins, and mitochondrial DNA have been proposed to contribute to aging (Karanjawala and Lieber, 2004). The reader is referred to the review by Balaban et al. in this issue of Cell concerning the potential role of mitochondrial DNA damage in aging (Balaban et al., 2005). Nuclear DNA is an attractive target for agingrelated changes since it must last the lifetime of the cell, unlike other cellular constituents, which can be replaced. In addition, the nuclear genome is present at only two to four copies per cell, rendering it potentially very vulnerable to lesions; by contrast, the mitochondrial genome is present at several thousand cop*Correspondence: alt@enders.tch.harvard.edu

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Figure 1. Multiple Pathways to Senescence These pathways are employed in a cell- and organism-specific fashion. In MEFs, activation of p19ARF leads to stabilization of p53. In certain human cell types (e.g., fibroblasts and keratinocytes), telomere attrition can activate p53 via the ATM and (potentially) ATR kinases. Senescence can also be triggered in human cells via p16 expression (Itahana et al., 2004). Activated p53 induces senescence via a complex gene expression program that includes induction of p21. The role of Rb in senescence involves repression of E2F target genes as well as alterations in chromatin structure. Senescent cells may contribute to aging via depletion of stem cell pools and/ or elaboration of factors that interfere with tissue function. Factors elaborated by senescent cells may also stimulate the growth of epithelial tumors (Krtolica and Campisi, 2002).

cent study of postmortem human brain tissue (Lu et al., 2004), which found that many nuclear genes involved in critical neural functions show reduced expression after age 40, concomitant with elevated levels of oxo8dG and DNA damage in their promoters. However, the high levels of oxidative DNA damage found by these investigators is at odds with other much lower estimates of the amount of oxidatively damaged DNA in cells (Hamilton et al., 2001). This highlights the difficulty of accurately measuring ROS and oxidative damage experimentally. Additional unresolved issues concerning the ROS theory of aging exist that will require clarification. If ROS are indeed an important source of aging-associated damage, it is currently unclear whether nuclear or mitochondrial DNA is the most relevant functional target (Barja and Herrero, 2000; Hamilton et al., 2001). Among long-lived mutants, the correlation with oxidative stress resistance is a frequent but not universal one: in some mutants, longevity occurs despite unchanged resistance to ROS or other forms of stress. Additionally, mice heterozygous for a mutation in the mitochondrial enzyme that processes superoxide, Sod2, live out a normal lifespan, despite accumulating higher levels of nuclear and mitochondrial 8oxodG with age (Van Remmen et al., 2003). Thus, while several lines of evidence argue for important role for ROS in aging, many questions remain regarding this hypothesis. In this regard, it is possible that additional mutations may be required to fully unveil potential effects of ROS (see below). Cellular Senescence: A Link between Cellular Damage and Aging? ROS and many other DNA-damaging agents can cause cells to enter a state of irreversible cell cycle arrest accompanied by characteristic morphologic and functional alterations, termed senescence (Ben-Porath and Weinberg, 2004). The induction of senescence depends on pathways involving the p53 and Rb proteins (Figure 1). Cellular senescence has been best characterized in cultures of human fibroblasts and mouse embryo fibroblasts (MEFs), which cease expanding after repeated

passage in culture, a process termed replicative senescence. Replicative senescence has been employed as a cellular model for aging; many mutations in DNA repair genes that cause premature aging phenotypes also confer premature replicative senescence (Table 1). Replicative Senescence Differs between Human and Mouse Cells In many primary human cell lines, replicative senescence occurs secondary to attrition of the ends of the chromosomes, the telomeres, in a process termed intrinsic senescence (Itahana et al., 2004). In mammalian cells, the ends of the chromosomes consist of a terminal 3# single-stranded tail, the G strand overhang, which is buried into adjacent double-stranded repetitive telomeric DNA, forming a protective t loop higherorder structure (de Lange, 2002). This t loop is stabilized by a D loop, or displacement loop: the region formed between the invading end of the telomere into adjacent double-stranded DNA. The G strand overhang is the substrate for the enzyme telomerase, which employs an RNA template, Terc, to extend telomeres during S phase, thereby countering the natural shortening of telomeres that would otherwise occur with each cell division. Telomerase access to its substrate is, in turn, regulated by telomeric proteins, which also modulate the conformational changes required for telomere replication and subsequent reestablishment of a protective end structure. In many types of human cells, including fibroblasts, telomeres shorten with each successive generation. Critically short telomeres trigger the onset of senescence through a process that may involve loss of the t loop structure and/or loss of protective proteins (referred to as uncapping). Such uncapped telomeres are then recognized by the cell cycle checkpoint machinery as DNA damage, leading to cell cycle arrest (Ben-Porath and Weinberg, 2004). In contrast to primary human fibroblasts, which express very low levels of telomerase activity, MEFs derived from M. musculus possess long telomeres and easily detectable telomerase activity (Itahana et al., 2004). Thus, MEFs ordinarily do not senesce due to telomeric attrition. Instead, senescence of wild-type

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Table 1. Features of Selected Models of Premature Aging in the Mouse Accelerated Fibroblast Senescence? Yes

Mutant Atm

Cellular Process Affected DSB signaling/repair

Tissues Affected Cerebellum, Gonad, Hematopoietic organs, Thymus Bone, Lens, Skin, Gonad Bone, Eye, Heart, Intestine, Liver, Lymphocytic hyperplasia, Testes +others Bone, Intestine Liver (Ercc1, XPF) + Brain, Kidney, Skin, SpleenErcc1 Bone, Liver, Skin Bone, Hair, Lymphoid tissue, Skin Bone, Testes Bone, Hair, Kidney, Skin, Thymus Bone, Hair, Heart, Hematopoietic organs, Skin, Testes Hematopoietic organs, Testes Hematopoietic organs, Hair, Heart, Intestine, Myometrium, Skin, Testes Bone, Brain, Hair, Hematopoietic organs, Intestine Intestine, Various Various Intestine, Various Bone, Endocrine, Gonad, Hair, Intestine, Lens, Skin, Spleen Bone, Endocrine, Gonad, Hair, Intestine Kidney, Lymphocytic infiltrates, Pancreatic islets, Skin, Testes None

Increased Rate of Cancer? Yes

Citations (Ito et al., 2004; Shiloh and Kastan, 2001) (Baker et al., 2004) (Cao et al., 2003b)

Bub1bH/H BRCA111/11/ p53+/

Spindle assembly checkpoint DSB repair, other

No Yes

Yes Yes

DNA-PKcs Ercc1, XPF

NHEJ, other Nucleotide excision repair, crosslink repair, other NHEJ, other DNA damage response DNA damage response DNA methylation Mitochondrial DNA polymerase

Yes No

No Yes (Ercc1)

Ku80 Dysfunctional p53 (p53m/+) Dysfunctional p53 (p44 Tg) PASG/Lsh PolgA

No No No No No

Yes ND Yes Yes ND

(Espejel et al., 2004b) (McWhir et al., 1993; Tian et al., 2004; Weeda et al., 1997) (Vogel et al., 1999) (Tyner et al., 2002) (Maier et al., 2004) (Sun et al., 2004) (Trifunovic et al., 2004)

Rad50S/S Terc

DSB repair Telomere maintenance

Yes Conflicting results

No Yes

(Bender et al., 2002) (Espejel et al., 2004a; Lee et al., 1998)

Terc/Atm

Telomere maintenance/ DSB signaling/repair Telomere maintenance/ NHEJ, other Telomere maintenance/ DNA repair Telomere maintenance/ NHEJ, other Telomere maintenance/ DNA repair

No

Yes

(Wong et al., 2003)

Terc/DNA-PKcs Terc/Parp-1 Terc/Ku80 Terc/Wrn

No No No Yes

ND ND ND Yes

(Espejel et al., 2004a) (Espejel et al., 2004a) (Espejel et al., 2004a) (Chang et al., 2004)

Terc/Wrn/Blm

Telomere maintenance/ DNA repair Topoisomerase

Yes

Yes

(Du et al., 2004)

TopIIIbeta

No

ND

(Kwan et al., 2003)

Wrn

DNA repair

No

Yes

XPA/CSB XpdTTD XpdTTD/XPA

NER, transcription NER, transcription NER, transcription

Cerebellum Bone, Hair, Ovary, Skin Bone, Hair, Skin

No Yes No

ND ND ND

(Lebel and Leder, 1998; Lombard et al., 2000) (Murai et al., 2001) (de Boer et al., 2002) (de Boer et al., 2002)

MEFs occurs primarily in response to oxidative DNA damage incurred during cell culture (Parrinello et al., 2003) in a process termed extrinsic senescence (Itahana et al., 2004). These differences between human and mouse cells with respect to senescence are not as

great as they may seem. Terc deficiency in mice leads to progressive telomere attrition in successive mouse generations and rapid senescence in embryonic fibroblasts derived from late-generation animals (Espejel and Blasco, 2002). Replicative life span of human cells

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is altered by culture under different oxygen tensions, and telomere shortening in these cells is itself accelerated by elevated levels of oxidative stress. It is now clear that senescence can be induced by a variety of different types of cellular injury (Figure 1). Cellular Senescence and Aging What is the relationship between cellular senescence and aging? The evidence that cellular senescence actually plays a role in aging is correlative: senescent cells accumulate in vivo in mammals with increasing age and at sites of pathology (Itahana et al., 2004), and many mouse and human models of premature aging are accompanied by premature cellular senescence in vitro (Table 1). Of note, late-generation Terc-deficient mice show some signs of accelerated aging (Lee et al., 1998; Rudolph et al., 1999). Two general models have been proposed to explain how cellular senescence may contribute to aging (Krtolica and Campisi, 2002; Pelicci, 2004). First, senescence of progenitor or stem cells themselves could impair tissue renewal. In this regard, the Polycomb group repressor Bmi1 appears to control levels of hematopoietic stem cells via negatively regulating the induction of senescence specifically in these stem cells. Second, senescent cells secrete proteases and other factors that may disrupt tissue function. In this regard, senescence has a complex relationship with neoplasia. Senescence has been postulated to occur as a tumor suppressor mechanism, whereby cells that have undergone a genotoxic insult and therefore possess the potential for neoplastic transformation enter a state in which they are incapable of dividing (Krtolica and Campisi, 2002). However, senescent stromal cells can actually promote the growth of epithelial cancers, malignancies that occur with increased incidence in the elderly. Senescence has been offered as an example of antagonistic pleiotropy, a process that is beneficial in young organisms but deleterious later in life: senescence suppresses cancer by preventing potentially tumorigenic cells from dividing but may potentially contribute to organ dysfunction in the aged through a variety of mechanisms, perhaps even contributing to neoplasia in this setting (Krtolica and Campisi, 2002). However, a causal relationship between cellular senescence and organismal aging has yet to be proved. DNA Repair and Aging The accurate maintenance of nuclear DNA is critical to cellular and organismal function, and therefore, numerous DNA repair systems have evolved. There is some evidence that the intrinsic fidelity and activity of such systems in different species may influence the rate of age-associated functional decline (Hart and Setlow, 1974), although such studies need to be performed with modern methodology. As outlined below, the efficiency of cellular DNA repair machinery itself may decline with age. Many different types of DNA lesions exist. DNA DSBs are repaired via the nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, whereas lesions on a single strand of DNA are repaired via base excision repair (BER) and nucleotide excision repair (NER) (and its subpathways). In mice and humans, mutations in certain DNA repair genes lead to

Table 2. Common Pathologic Features of Aging in Mice (after Bronson and Lipman [1991]; Cao et al. [2003a]) Hyperplasia/Neoplasia Adrenal hyperplasia Angiosarcoma Harderian gland adenoma Endometrial hyperplasia Lung adenoma Lymphoma Mammary gland adenocarcinoma Mast cell tumor Ovarian cystadenoma Paraovarian cyst Pituitary adenoma Sarcoma Thyroid follicular cell hyperplasia Uterine leiomyoma/leiomyosarcoma Leukocytic Infiltrates Kidney Liver Lung Mesentery/omentum Perineurium Salivary gland Genitourinary system Hydronephrosis Ovarian/testicular atrophy Seminal vesicle dilation Renal tubular dilation Bone Decreased cancellous bone Degenerative joint disease Molar teeth periodontitis Proliferations in the head/spine Neurological Hydrocephalus Neuronal lipofuscinosis Radiculopathy White matter gliosis Other Amyloidosis Fatty change of the liver Focal myocardial degeneration Hepatocyte polyploidization Thymic involution

phenotypes that, in some respects, caricature aging. For reference, the features of aging as it occurs in wildtype mice are enumerated in Table 2, and the features of mouse DNA repair/metabolism mutants showing some features of aging are listed in Table 1. We now turn to discussing specific gene defects and their relevance to aging. The ATM-p53 Axis: A Link between the DNA Damage Response and Aging? Unrepaired or improperly repaired DSBs have serious potential consequences for the cell: cell death, senescence, dysregulation of cellular functions, genomic instability, and, in higher organisms, oncogenic transformation. The initial step in DSB repair is detection of the lesion, and this step involves the ataxia-telangiecta-

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Figure 2. A Highly Simplified View of the DNA Damage Response See text for details.

sia mutated (ATM) kinase, other related phosphatidylinositol 3-kinase-like kinases (PIKKs), and other proteins (Figure 2; Bassing and Alt, 2004). Activated ATM phosphorylates numerous proteins involved in the G1/S, intra-S, and G2/M checkpoint responses and additionally phosphorylates factors involved in DNA repair (Bassing and Alt, 2004). An important target of ATM is the p53 protein, which plays a role in the cellular response to numerous genotoxic insults including DSBs. Phosphorylation of p53 by ATM and kinases downstream of ATM is a major mechanism leading to upregulation of p53 levels and activity, although p53 also can be activated via ATM-independent mechanisms. Activated p53 stimulates or represses transcription of many target genes and coordinates checkpoint, senescence, and apoptosis pathways in response to DSBs and other signals. In this regard, p53, through modulation of various downstream targets, triggers arrest/senescence or apoptosis (Meek, 2004). The exact factors that determine the differential outcomes of this complex program are not yet completely elucidated but vary with the cell type, as well as the kind, intensity, and duration of the damage. ATM and Aging Perturbations in ATM function can lead to symptoms of accelerated aging. Patients with mutations in the ATM gene suffer from Ataxia-Telangiectasia (AT), a condition characterized by a prematurely aged (progeroid) appearance, immunodeficiency, cerebellar degeneration, and cancer (Shiloh and Kastan, 2001). ATM deficiency in mice recapitulates many of these phenotypes, although the progeroid features of the mouse models are less prominent than in the human disease. Given the many targets of ATM, it is difficult to trace the progeroid appearance of AT patients to a specific function of this

protein although several possible mechanisms are conceivable (see below). In this context, the phenotype of ATM-deficient cells may be relevant. In particular, such cells manifest sensitivity to DSB-inducing agents and have marked genomic instability; they also grow poorly and senesce prematurely in culture (Barzilai et al., 2002). Genomic instability or DNA repair defects of ATM deficiency could contribute to the aging-like features of this disorder (see below). In this context, the premature cellular senescence phenotype of ATM deficiency is rescued by p53 deficiency (Xu et al., 1998), suggesting a role for ATM-independent, p53-dependent checkpoint responses. By extension, such responses also might contribute to premature-aging phenotypes associated with ATM deficiency. Another potential function for ATM in suppressing progeroid phenotypes may be related to reported ATM functions in regulating intracellular ROS levels and sensitivity to these molecules (Barzilai et al., 2002; Ito et al., 2004). Persistently elevated ROS levels in AT cells might cause chronic damage to DNA and other cellular macromolecules. However, given the lack of an AT-like phenotype of the Sod2+/ mouse, progeroid features of ATM deficiency likely reflect more than increased ROS levels. One possibility would be elevated ROS levels in conjunction with loss of another ATM function, such as ability to respond to ROS-induced DNA damage. Finally, ATM plays an important role in telomere maintenance; AT cells show shortened telomeres and an increased incidence of telomeric fusions (Pandita, 2002). Moreover, mice lacking both ATM and Terc have higher levels of telomeric dysfunction than generation-matched Terc-deficient mutants, as well as proliferative defects in multiple tissues, decreased survival, and clinical evidence of premature aging (Wong et al., 2003). Thus, human AT patients may show aging-like effects, at least in part, as a consequence of telomeric dysfunction, an effect that ordinarily may be masked in the mouse due to the long telomeres of this organism. The phenotypes of ATM deficiency are influenced by environmental conditions. In the ATM-deficient mouse, the onset of T cell lymphoma can be delayed by treatment with an antioxidant, suggesting that dietary factors impact on the expression of this condition (Schubert et al., 2004). Neurological dysfunction in this model can similarly be ameliorated via antioxidant treatment (Browne et al., 2004). Additionally, the frequency of thymic lymphoma varies among different strains of ATM-deficient mice, a finding that might reflect different background mutations and/or housing conditions and the types of pathogens present (Petiniot et al., 2002). These observations raise the possibility that other aspects of the ATM-deficient phenotype, including the premature aging observed in human patients, may be influenced by environment. This is a theme that we will return to in our discussion of other premature aging models. p53 and Aging Various lines of evidence suggest that p53 plays opposing roles in the aging process. While p53 suppresses the onset of malignancy and thereby extends life span, at the same time it promotes cellular senescence and apoptosis in response to DNA damage, potentially contributing to the clinical changes of aging.

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Figure 3. NHEJ at General DSBs Factors associated with progeroid mutant phenotypes (Ku80 and DNA-PKcs) are shown in red. See text for details.

Thus, p53 function may display antagonistic pleiotropy (Campisi, 2002). The role of p53 in aging cannot be directly tested using p53-deficient mice, as such animals invariably die of malignancy before age-related changes become manifest. However, two mouse strains that express C-terminal p53 fragments along with full-length p53 have been reported to show accelerated aging phenotypes and a lower incidence of malignancy (Maier et al., 2004; Tyner et al., 2002). It has been proposed that these truncated p53 proteins exert their effects by modifying the activity of endogenous wild-type p53 protein. Only nonphysiologic activation of p53 leads to progeria, as mice expressing extra copies of wild-type p53 under the control of its own promoter do not show signs of premature aging (Garcia-Cao et al., 2002). Further arguing for a role for p53 in promoting aging, a null mutation in a gene functioning downstream of p53 in the induction of apoptosis, p66Shc, confers oxidative stress resistance and extends mouse life span (Migliaccio et al., 1999). The p66Shc protein shortens murine life span via at least two mechanisms: it increases constitutive intracellular ROS levels, and it promotes cell death in response to oxidative stress. It is unclear how p66shc evolved to play such a role in the cell, since the shorter isoforms of this protein, p52 and p46, fulfill an entirely dissimilar cellular function, transducing signals from tyrosine kinases to ras. A Putative Role for NHEJ in Suppressing Aging NHEJ in DSB Repair and Telomere Maintenance We now turn from a discussion of factors upstream of DNA repair to a consideration of repair pathways themselves and their involvement in aging. NHEJ is one of the two major DSB repair pathways in mammalian cells (Bassing and Alt, 2004). NHEJ is mediated by at least six core factors (Figure 3). Four of these proteins (Ku80,

Ku70, Ligase IV, and XRCC4) are conserved from yeast to mammals; they are indispensable for all NHEJ reactions. In contrast, the other two NHEJ factors, DNAPKcs (DNA-dependent protein kinase catalytic subunit) and Artemis, have evolved more recently and are thought to be required for joining the subset of DNA ends that require processing prior to ligation. Ku70 and Ku80 bind as a heterodimer to DSBs, where they are thought to serve a protective function and to enlist other factors. In this regard, Ku70 and Ku80 recruit DNA-PKcs, and together the three proteins form the DNA-PK holoenzyme. DNA-PK activates Artemis, which functions as an endonuclease to process ends that cannot be directly rejoined. Finally, XRCC4 and Ligase IV, which are likely recruited by Ku, function together to catalyze end ligation itself. NHEJ often occurs concomitant with loss of a few nucleotides at the site of joining. NHEJ plays a critical role in general DNA DSB repair and, correspondingly, in the maintenance of genomic stability. In addition, NHEJ plays a role in repairing genetically programmed DSBs in the context of antigen receptor variable region gene assembly (V(D)J recombination) in developing lymphocytes. Mice with targeted inactivating mutations in NHEJ genes display phenotypes that reflect loss of these functions (Ferguson and Alt, 2001). All NHEJ-deficient mice suffer from severe combined immunodeficiency as a consequence of an inability to productively rejoin broken V(D)J gene segments in developing B and T cells. NHEJ deficiency is also associated with ionizing radiation-sensitivity and an elevated incidence of spontaneous genomic instability. MEFs deficient for XRCC4, Ligase IV, Ku70, or Ku80 (but not DNA-PKcs or Artemis) senesce prematurely in culture, and mice deficient for these four factors are very small and show widespread neuronal apoptosis during embryogenesis. Premature senescence and neuronal apoptosis, but not small size, are

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relieved by p53 deficiency. Thus, the former phenotypes occur as a response to, rather than as a direct consequence of, unrepaired DSBs (Ferguson and Alt, 2001). Mice deficient in NHEJ and p53 invariably succumb to pro-B cell lymphomas as a consequence of aberrant repair of V(D)J recombination-associated DSBs in developing B cells, leading to oncogenic translocations (Bassing and Alt, 2004). In addition to their roles in DSB repair, at least three core NHEJ factors, namely Ku70, Ku80, and DNA-PKcs, localize to telomeres (d'Adda di Fagagna et al., 2001). Deficiency for any of these, as well as for Artemis (Rooney et al., 2003), is associated with an increased frequency of end-to-end chromosomal fusions in MEFs, suggesting a role for these proteins in chromosomal end capping. There is conflicting data on whether NHEJ factors protect against telomere shortening. NHEJ also promotes telomeric fusions in some circumstances. Increased telomere end-to-end fusions are observed in the setting of the telomeric attrition associated with Terc deficiency or inhibition of TRF2, a protein thought to function in end capping, and these end-to-end fusions are eliminated in NHEJ-deficient backgrounds (Espejel et al., 2002a, 2002b; Smogorzewska et al., 2002). Thus, by ligating uncapped telomeres, NHEJ may actually promote genomic instability. Aside from roles in the NHEJ reaction and in telomere maintenance, Ku70, Ku80, DNA-PKcs, and Artemis have other known or suspected cellular functions, including DSB or checkpoint signaling, whereas XRCC4 and Ligase IV appear to function only in end-ligation during NHEJ. In summary, NHEJ plays crucial roles in general and site-specific DSB repair, in telomere maintenance, and in maintenance of genomic stability. A Potential Relationship between Ku80 Deficiency, DNA-PKcs Deficiency, and Aging NHEJ has been proposed to play a causative role in the aging process. DSBs are frequent events in mammalian somatic cells, where they are also very commonly repaired by NHEJ. In addition, genetic studies support the notion that NHEJ plays an important role in the repair of ROS-induced DNA lesions (Karanjawala and Lieber, 2004). Since NHEJ can delete a few nucleotides at sites of DSB repair, this process theoretically could lead to accumulation of mutations and contribute to cellular decline and aging (Karanjawala and Lieber, 2004). Moreover, in the absence of NHEJ, DSBs often are repaired concomitant with large deletions and/or translocations; thus, absent or even decreased levels of NHEJ also might contribute to accelerated aging. The observation that phenotypes resembling accelerated aging have been described in one strain each of Ku80- and DNA-PKcs-deficient mice potentially supports this model. Thus, a line of Ku80-deficient mice prematurely exhibits age-specific changes including osteopenia, atrophic skin, liver lesions, and shortened life span (Vogel et al., 1999). Likewise, a strain of DNAPKcs-deficient mice recently has been noted to exhibit age-related pathologies, with osteopenia, intestinal atrophy, thymic lymphoma, and reduced longevity (Espejel et al., 2004b). There are several potential explanations to account for how NHEJ deficiency could cause aging-like phenotypes. As described above, spontaneous DSBs in Ku80- or DNA-PKcs-deficient animals

could directly impair cellular function by promoting DNA deletions. In this context, Ku80-deficient mice are found to have a low rate of mutations at a marker locus (Rockwood et al., 2003); such an assay might not detect large deletions or rearrangements, however. Alternatively, unrepaired DSBs could trigger elevated levels of cellular senescence and apoptosis. In this regard, p53-dependent responses are critical in mediating the premature senescence and neuronal apoptosis phenotypes of certain NHEJ mutants (Ferguson and Alt, 2001). Despite the phenotypes of the Ku80- and DNA-PKcsdeficient mice, several observations suggest that any potential roles for NHEJ in suppressing aging might be more complicated. Ku70- and Artemis-deficient mice thus far have not been reported to show premature aging phenotypes. If Ku70-deficient mice actually lack an aging phenotype, the apparent Ku80-deficient progeroid phenotype must be rationalized in the context of the finding that targeted ablation of Ku70 results in dramatically reduced Ku80 levels (Gu et al., 1997). In addition, aging-related phenotypes thus far have not been reported in several other independently generated DNA-PKcs mutant mouse strains. Ligase IV- and XRCC4deficient mice show embryonic lethality due to widespread neuronal apoptosis, precluding aging analyses. However, the lack of a consistent aging-like phenotype in other lines of long-lived Ku-, DNA-PKcs-, or Artemisdeficient mice is difficult to explain if NHEJ plays a general role in delaying manifestations of aging (Karanjawala and Lieber, 2004). In this regard, it is conceivable that some aging-related phenotypes might have been missed due to lack of thorough examination of other strains of NHEJ-deficient mice. Background mutations in various strains of NHEJdeficient mice, either exacerbating or suppressing the progeroid manifestations of NHEJ deficiencies, might contribute to the apparently discrepant phenotypes of different lines. This possibility, which is relevant to any gene deficiency/aging model, warrants further examination and is discussed in other contexts below. Also, given that both the Ku80- and DNA-PKcs-deficient mice show evidence of ongoing inflammation, potentially indicative of infection, it is conceivable that some aging-like phenotypes might occur directly due to the effects of chronic infection in the setting of immunodeficiency, rather than due to impaired DNA repair. Osteopenia, for example, could result from elevated glucocorticoid levels induced by chronic physiologic stress from infection or malnutrition associated with intestinal atrophy. The degenerative changes in afflicted strains of DNA-PKcs- and Ku80-deficient mice affect only a limited subset of organs. Therefore, either these NHEJ factors are only involved in suppressing aging-related changes in certain tissues or these degenerative changes occur for reasons distinct from those that contribute to aging in wild-type animals. Of note, degenerative changes in these models occur in both highly proliferative (intestine and skin) and relatively less proliferative (bone and liver) tissues, apparently sparing many other tissues of both types. These observations argue against a simple relationship between mitotic status and dependence on NHEJ in the suppression of aging. In addition to the above considerations, other models

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for NHEJ factor functions in suppressing aging may be postulated. It is notable that efficiency of DSB repair may decline with age in budding yeast (McMurray and Gottschling, 2003) and in mammalian tissues (Sedelnikova et al., 2004; Singh et al., 2001), as well as in senescent mammalian cells (Seluanov et al., 2004). Theoretically, such an age-related decline in DSB repair might contribute to increased mutations and genomic rearrangements preferentially near the end of life, although such a decline has not been rigorously proven. Also, loss of other functions of DNA-PKcs or Ku80, such as telomere end capping and/or DNA damage signaling, could contribute to aging phenotypes. In this context, targeted inactivation of either Ku80 or DNAPKcs, but not in Ligase IV or XRCC4, demonstrates synthetic lethality with ATM deficiency. These observations argue for overlapping functions between Ku/DNAPKcs and ATM that do not involve classical NHEJ, perhaps related to telomere maintenance (Sekiguchi et al., 2001). Moreover, Ku80/Terc and DNA-PKcs/Terc double deficient mice show exacerbation of intestinal atrophy and other aging-like phenotypes of Terc deficiency (Espejel et al., 2004a). Late generation Terc-deficient mice have an uncharacterized DSB repair defect (Wong et al., 2000); loss of NHEJ in the context of dysfunctional telomeres would be predicted to further compromise DSB repair. In this regard, cells deficient in Ligase IV and ATM or Terc and ATM show high levels of genomic instability and very rapid senescence (Sekiguchi et al., 2001; Wong et al., 2003), pointing to synergies between DSB repair, DNA damage signaling, and telomere maintenance in genomic maintenance and overall cellular viability. In summary, there are many potential roles for NHEJ in preventing premature aging-like phenotypes, such as those observed in Ku80- and DNA-PKcs-deficient mice. Current findings suggest, however, that aging phenotypes likely do not result from a failure of DSB repair alone but instead from a loss of other functions or combinations of functions of these proteins, perhaps in concert with environmental factors, such as housing conditions and/or infection and/or background mutations. Such complexities also may likely apply to other DNA repair proteins implicated in the suppression of aging as well, since, as we shall see, many of these proteins play roles in other cellular processes besides DNA repair and, as with NHEJ, only a subset of mutants in any given DNA repair pathway show aging-related phenotypes. Potential Roles for Rad50 and BRCA1 in HR and Aging HR in DSB Repair Two factors with roles in HR, Rad50 and BRCA1 (breast cancer susceptibility gene-1), appear linked to aginglike phenotypes in mouse models. HR is the other major pathway of DSB repair in mammalian cells. Unlike NHEJ, HR uses the sister or (in some cases) the homologous chromosome as a template to repair the broken chromosome. Whereas recent data indicate that NHEJ functions in DSB repair throughout the cell cycle, HR is largely restricted to late S/G2 (Couedel et al., 2004; Mills et al.,

2004; Rothkamm et al., 2003; Takata et al., 1998). The first step in HR is processing of DSBs by a nuclease to generate 3# ssDNA tails, which are coated with RPA protein (Figure 4). The MRN complex, composed of the proteins Mre11, Rad50, and Nbs1, is a candidate for this nuclease, although other nucleases are likely involved as well. The MRN complex plays multiple other roles in DSB repair: it functions both upstream and downstream of ATM and has been proposed to promote sister chromatid association and recombination (Stracker et al., 2004). The Rad51 protein, assisted by a number of factors including Rad52, Rad54, BRCA2, and the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D), forms a nucleoprotein complex with the DNA and directs the 3# ssDNA tails to search out, invade, and pair with undamaged homologous sequences. DNA polymerases then carry out repair using the intact DNA as a template. The processes of DNA strand exchange and extension generate Holliday junctions (HJs), structures in which two dsDNA duplexes are intertwined. In mammalian DSB repair, HJs are thought to be resolved primarily via disengagement and gap repair rather than cleavage of the HJ (Valerie and Povirk, 2003). BRCA1 in HR and Other Cellular Processes The biology of the BRCA1 protein has proven to be very complex; BRCA1 plays roles in multiple fundamental cellular processes. Biochemically, BRCA1, together with its partner protein BARD1, possesses E3 ubiquitin ligase activity in addition to binding both DNA and multiple other proteins. Functionally, several lines of evidence link the BRCA1 protein to HR (Scully et al., 2004). BRCA1 is a phosphorylation target of ATM and the related PIKK, ATR, following DNA damage induction. BRCA1 forms a complex with Rad51 and BRCA2 (Dong et al., 2003) and can be detected with Rad51 in S phase foci thought to represent stalled replication forks. BRCA1 foci also form in response to ionizing radiation and on chromosomes during meiotic recombination. BRCA1 deficiency leads to impaired HR-mediated repair of chromosomal DSBs, hypersensitivity to many DNA-damaging agents, and genomic instability. The biochemical nature of BRCA1s involvement in HR remains unclear; one possibility is that BRCA1 may perform a scaffolding function, potentially coordinating the formation of functional repair complexes at DSBs. The E3 ubiquitin ligase activity of BRCA1 may also be relevant in this context; BRCA1 may modify other proteins during HR to alter their functions or direct their degradation. Additionally, BRCA1 has been implicated in many other functions outside HR: transcription, G2/M checkpoint control, chromatin remodeling, and X inactivation (Scully et al., 2004). It has also been proposed that BRCA1 may play a role in NHEJ (Ting and Lee, 2004). Thus, BRCA1 plays roles in numerous cellular processes, including DNA repair. BRCA1 and Rad50 Hypomorphs Show Aging Phenotypes Defects in Rad50 or BRCA1 cause progeroid phenotypes. Deficiency of Mre11, Nbs1, or Rad50 is not compatible with cellular survival (Stracker et al., 2004). However, homozygosity for a Rad50 hypomorphic allele (Rad50s/s) permits viability; Rad50s/s mice show a shortened life span, cancer predisposition, and hemato-

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Figure 4. HR at DSBs See text for details. Reproduced by permission with modification from Oncogene (Valerie and Povirk, 2003), copyright 2003 Macmillan Publishers Ltd.

poietic stem cell and spermatogenic failure (Bender et al., 2002). Genomic instability is detectable in cells derived from this animal. The attrition of the hematopoietic and male germ cell lineages occurs in large measure due to p53-mediated signaling triggered by genomic instability (Bender et al., 2002). Homozyogous inactivation of BRCA1 results in early embryonic lethality (Valerie and Povirk, 2003); however, mice homozygous for a BRCA1 hypomorphic allele and haploinsufficient for p53 (BRCA111/11/p53+/) are viable and have many features reminiscent of accelerated aging: wasting, skin atrophy, osteopenia, and malignancy (Cao et al., 2003b). There is compelling evidence that this phenotype results from p53-dependent responses to unrepaired DNA damage (Cao et al., 2003b). Baseline p53 protein levels are higher in BRCA111/11/ p53+/ mice than in p53+/ control animals. Similar to some NHEJ-deficient MEFs, BRCA111/11 MEFs show increased chromosomal abnormalities and premature cellular senescence, and BRCA111/11 embryos show tissue SA--galactosidase activity, a marker of senescence. Thus, in both the Rad50s/s and BRCA111/11/ p53+/ mice, signs of premature aging occur as a consequence of the p53-mediated responses to unrepaired DNA damage. However, since BRCA1 and Rad50 are both involved in multiple cellular processes, the possibility exists that loss of other functions beyond their

DNA repair roles may contribute to these aging-like mutant phenotypes. Single-Stranded DNA Lesions and Aging Nucleotide Excision Repair DNA lesions that affect only one DNA strand are repaired via BER or NER and its subpathways. Although BER is thought to play a critical role in the repair of oxidative lesions, mutations in genes involved in this pathway do not produce aging manifestations: they are either lethal or confer no obvious phenotypes (Hasty et al., 2003). By contrast, lesions in some factors involved in NER can lead to premature aging syndromes in mice and humans. NER is activated by a wide range of helixdistorting DNA lesions, including UV-induced photoproducts, bulky chemical adducts, and certain oxidative lesions. NER can be subdivided into two pathways, global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which differ with respect to the lesion detected and some of the factors involved (Mitchell et al., 2003; Figure 5). The basic NER machinery consists of the proteins XPA through XPG, the CSA and CSB proteins, and other participants such as the basal transcription factor TFIIH. The GG-NER specific factors, XPC (in a complex with the HR23B protein) and XPE are responsible for detecting helix-distorting lesions that occur throughout

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Figure 5. Global-Genome NER and Transcription-Coupled NER Factors with aging-related mutant phenotypes (XPD, CSA, CSB, ERCC1, and XPF) are shown in red. See text and Table 1 for details. Reprinted with modification from Mitchell et al. (2003) with permission from Elsevier.

the genome. The TC-NER specific factors, CSA and CSB, are involved in repair specifically in transcribed regions and, when RNA polymerase II stalls at a lesion, contribute to displacing the stalled polymerase to create access for repair machinery. Following recognition of the damaged DNA, common NER factors are recruited in both GG-NER and TC-NER. TFIIH, which contains two DNA helicases, XPB and XPD, unwinds the DNA flanking the lesion. The single-strand DNA (ssDNA) binding protein RPA binds to and stabilizes the unwound DNA strands, and XPA aids in lesion recognition. Two structure-specific endonucleases, XPG and XPF (the latter in a complex with the protein ERCC1), then make single-strand incisions on either side of the lesion to release an oligonucleotide. The resulting gap is filled by template-dependent DNA polymerization followed by ligation. Several core NER factors have been implicated in processes aside from NER. XPB and XPD are critical in RNA polymerase II transcription; CSB associates with RNA polymerases I and II; and XPF/ERCC1 has been implicated in repair of interstrand crosslinks, homology directed repair, and processing of the 3# G strand overhang at telomeres. Some NER Defects Lead to Premature Aging Phenotypes Numerous human patients and mouse strains with defects in different NER factors exist, and some have phenotypes reminiscent of premature aging (Mitchell et al., 2003). Defects in the TC-NER-specific factors CSA or CSB lead to Cockayne syndrome in humans, a severely debilitating disorder with striking progeroid features. CSA- and CSB-deficient mice show much milder phenotypes than their human counterparts (Mitchell et al., 2003). Patients with specific mutations in XPD suffer from trichothiodystrophy (TTD), a disease characterized by photosensitivity, brittle hair, skin defects, and a shortened life span. These patients show defects in transcription of hair- and skin-specific transcripts (and perhaps other mRNAs) (Bergmann and Egly, 2001). In addition, cells derived from TTD patients show NER defects, suggesting that multiple functions of XPD are de-

fective in these individuals. Mice bearing a targeted mutation in XPD that recapitulates a human TTD mutation show similar manifestations to human TTD patients. Additionally, these XPDTTD mice show aging-associated changes such as wasting, scoliosis, osteoporosis, and melanocyte loss (de Boer et al., 2002). XPDTTD/XPA double mutants show a much more rapid degenerative phenotype, suggesting that in the setting of impaired transcription and/or TC-NER, a total lack of NER is extremely deleterious. Similarly, mice deficient in both CSB and XPA also die within a few weeks after birth, although only cerebellar defects have been described in detail in these animals (Murai et al., 2001). The aging-like phenotypes in human CS patients and in XPDTTD, XPDTTD/XPA, and CSB/XPA mouse mutants may be explained by the fact that a failure to repair lesions in transcribed genes can result in cell death, leading to tissue attrition and aging. Stalled RNA polymerase provides a signal for activation of p53-dependent apoptosis (Ljungman and Lane, 2004). In this regard, it will be of interest to determine whether p53 deficiency rescues the aging-like features of XPDTTD, XPDTTD/XPA, and CSB/XPA mice. Alternatively, the involvement of XPD and the CSB proteins in transcription suggests that impaired transcription of critical genes may play a role in causing these progeroid phenotypes, perhaps interacting with the repair defects in a complicated fashion. WRN and Aging Defects in proteins with less well-defined functions in DNA repair can lead to aging phenotypes as well. The human disease WS represents the best model for premature aging in humans (Goto, 1997). WS patients develop premature graying, cataract, loss of subcutaneous fat, skin atrophy, osteoporosis, diabetes, atherosclerosis, and malignancies. WS cells senesce prematurely in culture. The gene defective in WS, WRN, encodes a helicase of the RecQ family (a group defined by its similarity to E. coli RecQ helicase). WRN also possesses an exonuclease domain. WRN plays a role in the maintenance of overall genomic stability, and

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WRN may be involved in multiple DNA repair pathways (Bachrati and Hickson, 2003). Recent experiments using mice bearing targeted mutations in WRN provide evidence that, with respect to aging, the most relevant sites of WRN function are the telomeres. WRN-deficient mice do not recapitulate human WS (Bachrati and Hickson, 2003). Based on the observations that human WS cells show telomeric instability (Schulz et al., 1996; Tahara et al., 1997) and that introduction of telomerase into WS fibroblasts can rescue their premature senescence (Wyllie et al., 2000), it was proposed that WRN-deficient mice may not demonstrate a strong phenotype due to the abundant mouse telomere reserve (Lombard et al., 2000). This hypothesis has been proven correct by the generation of mice deficient in both WRN and Terc (Chang et al., 2004; Du et al., 2004). In the WRN/Terc double deficient animals, phenotypes reminiscent of human WS that are not observed in either single mutant are present: osteopenia, diabetes, and sarcomas. Phenotypes ordinarily seen in late-generation Terc-deficient animals also occur earlier in the compound mutants and are associated with a greater degree of telomeric dysfunction. Whether or not these phenotypes depend on p53 function is unknown; the premature senescence of human WS fibroblasts is p53 dependent (Davis et al., 2003). The latter observation suggests that cellular checkpoint functions may be involved in producing the clinical features of WS. The exact role of WRN in telomere maintenance is currently unclear. WRN can be detected at the telomeres in the absence of telomerase function in mammalian cells (Johnson et al., 2001; Opresko et al., 2004), and Sgs1p, the S. cerevisiae WRN homolog, is required for recombinational telomere maintenance in telomerase-deficient cells in yeast (Cohen and Sinclair, 2001; Huang et al., 2001; Johnson et al., 2001). WRN also interacts with the telomeric protein TRF2 and can unwind and degrade telomeric D loop structures. These observations suggest that WRN may play a role in providing telomeric access to other factors involved in telomere maintenance (Machwe et al., 2004; Opresko et al., 2004; Orren et al., 2002). Overall, the phenotype of the WRN/ Terc double knockout mouse argues that defective telomere maintenance is an important factor in producing the premature aging-like aspects of WS in humans, although it does not exclude a role for other functions of WRN at nontelomeric sites. Sir2: A Link between Metabolism, Genome Stability, and Life Span In the models discussed above, decreased genomic stability is associated with shortened life span. The Sir2 family of proteins provides an example in which increased genomic stability extends life span (Blander and Guarente, 2004). In S. cerevisiae, the chromatin regulatory factor Sir2 (silent information regulator-2) functions as an NAD-dependent histone deacetylase (Imai et al., 2000) to suppress recombination and turns off transcription at multiple genomic loci (Blander and Guarente, 2004). One metric of aging in yeast is the number of divisions that a single mother cell undergoes. The excision and replication of rDNA circles

is an important cause of mortality in yeast when life span is measured in this fashion (Sinclair and Guarente, 1997). Loss of Sir2 increases rDNA recombination and shortens life span; whereas an extra genomic copy of Sir2, which increases rDNA stability, extends life span (Kaeberlein et al., 1999). It has been proposed that Sir2 activity ties the energy status of the yeast cell to longevity (Blander and Guarente, 2004; Lin et al., 2000). When nutrients are scarce, yeast cells preferentially employ respiration rather than fermentation to generate ATP (Lin et al., 2002). This metabolic switch alters the metabolism of the cell, increasing the NAD/NADH ratio and/or decreasing levels of the Sir2 inhibitor nicotinamide, in turn activating Sir2 and increasing rDNA stability (reviewed in Blander and Guarente, [2004]). Strikingly, overexpression or pharmacologic activation of Sir2 in worms and flies also extends life span (Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001; Wood et al., 2004). Sir2-driven increased longevity in C. elegans requires the Daf-16 transcription factor (Tissenbaum and Guarente, 2001). Daf-16 is a critical mediator in the insulinlike signaling pathway, normally employed by worms to arrest as extremely long-lived larvae under unfavorable environmental conditions; certain mutations in this pathway confer longevity upon adult worms. The mechanisms by which Sir2 extends life span in flies are currently unclear. Sir2 family members may play a general role in mediating caloric restriction (Sohal and Weindruch, 1996), an intervention capable of extending life span in many different organisms from yeast to mammals (Cohen et al., 2004; Howitz et al., 2003; Lin et al., 2000; Rogina and Helfand, 2004; Wood et al., 2004), although in yeast the involvement of Sir2 in CR appears to be strain specific (Kaeberlein et al., 2004). In mammals, there are seven Sir2 family members, designated SIRT1SIRT7 (Frye, 2000); SIRT1 is the most highly related to S. cerevisiae Sir2. The role of SIRT1 in mammalian longevity has not yet been directly tested, since on a pure strain background SIRT1-deficient animals die very early as a consequence of multiple developmental defects (Cheng et al., 2003; McBurney et al., 2003). Unlike yeast Sir2, which has no known targets aside from histones, SIRT1 possesses a large and growing list of targets, some of which, including p53 and forkhead transcription factors (mammalian homologs of Daf-16), modulate cellular resistance to oxidative and genotoxic stress (Blander and Guarente, 2004). Additionally, SIRT1, like Sir2, has recently been shown to directly modify chromatin and silence transcription (Vaquero et al., 2004). It is now important to determine whether SIRT1, in addition to silencing transcription, also suppresses recombination and genomic instability via chromatin effects and if so, whether such an activity could be involved in regulating aging in mammals. SIRT1 conditional alleles may allow studies of the role of this protein in aging. The true mammalian functional ortholog of Sir2, if one exists, might also be a different mammalian Sir2 family member (or members) than SIRT1. SIRT2 and SIRT3 are unlikely to play this role, because these proteins are cytoplasmic and mitochondrial, respectively, rather than chromatin associated (Blander and Guarente, 2004). Thus far, no information has been forth-

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Figure 6. A Model for the Role of Unresolved DNA Lesions in Aging Unrepaired DNA lesions can activate the cell cycle checkpoint machinery, leading to senescence or apoptosis and subsequent cellular attrition and tissue dysfunction. Some types of unrepaired DNA damagelike DNA DSBscan trigger genomic instability, which can in turn lead to further DNA damage. In addition, unrepaired DNA damage can directly compromise cellular processes like transcription, an effect that could also impair tissue function. Such unrepaired DNA damage will be frequent in DNA repair mutants and lead to accelerated degenerative changes; in wild-type organisms unrepaired damage is infrequent, owing to efficient repair systems.

coming regarding the functions of the four remaining SIRTs, SIRT4SIRT7, but these remain candidates for proteins that may regulate longevity through genome stabilization. Conclusions The hypothesis that nuclear DNA, a critically important cellular constituent that cannot be replaced, is an important target of age-related change is supported by evidence that nuclear DNA damage and mutations accumulate with age. While ROS are likely to be one important source of this damage, there are numerous other cellular and environmental sources of damage, and the impact of such lesions may be enhanced by age-related compromise of DNA repair. In the latter context, most premature aging syndromes are caused by mutations in genes encoding proteins involved in DNA repair (Karanjawala and Lieber, 2004). Accumulation of mutations in critical genes may be one general mechanism by which compromised DNA repair could contribute to aging. In addition, p53-mediated senescence and apoptosis, in response to DNA damage, also likely contribute to aging (Figure 6). Indeed, the fact that lesions in several disparate repair systems cause phenotypes that are broadly similar to one another (Table 1) is consistent with the notion that the specific chemical nature of the accumulated DNA lesions may be less important than their ability to activate the common cellular checkpoint machinery. It remains unclear why only certain DNA repair mutants in particular pathways show progeroid phenotypes. In some cases, it may simply be that some mutants have not been scrutinized sufficiently to reveal such effects. However, genetic background effects almost certainly play an important role in modifying the aging-like manifestations of DNA repair deficiencies. Also, it must be remembered that many of the DNA repair genes and factors implicated in suppressing aging also play roles in cellular processes other than DNA repair, and therefore aging-like phenotypes might be enhanced by impairment of other cellular functions in conjunction with altered DNA repair. In addition, as dis-

cussed above, environmental factors including housing conditions, infectious agents, diet, and many other influences, also likely play a significant role in the expression of aging phenotypes in mouse models and, perhaps, in humans as well. Thus, aging is likely the outcome of a complex interplay between the genetic endowment of an organism and the stresses placed upon it by its particular environment. All mouse models that link DNA repair to aging possess defects in DNA repair and have shortened life spans. It is important to bear in mind the potential pitfalls of such models (Hasty and Vijg, 2004; Miller, 2004). Aging encompasses a wide spectrum of degenerative processes, many of which are quite nonspecific, both clinically and pathologically (Harrison, 1994). Thus, it is difficult to arrive at a strict, experimentally useful definition of aging. Factors implicated in organismal decline in genetic models might not play a role in the normal aging processes. A related difficulty is that premature aging models fail to recapitulate all aspects of aging but are instead segmental progerias (Hasty and Vijg, 2004; Miller, 2004); that is, they reproduce in an accelerated fashion some but not all aspects of aging as it occurs in wild-type animals. In this regard, the myriad histopathologic changes of normal aging (Table 2) correspond poorly with the changes that occur in models of premature aging (Table 1). Mammalian aging is not likely a single process but rather the decline of many somatic functions, heavily influenced by the environment; this is a complex interplay that is extremely difficult to model accurately. For these reasons, genetic models of extended life span are likely to be more informative than models with reduced longevity with respect to physiologically relevant causes of decline and mortality; in such long-lived organisms, life span-limiting factors must of necessity be counteracted. There are many outstanding questions regarding the connection between DNA repair and aging that will benefit from the application of emerging techniques in molecular biology and genetics. In models of premature aging, the most vulnerable system fails first, leading to death and precluding gain of insights from effects on other, potentially more relevant, organ systems. This

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difficulty might be overcome with hypomorphic alleles or via conditional knockouts of relevant genes. The latter approach, for example, might allow an evaluation of the roles of genes essential during embryogenesis in the aging process. Such an approach also could permit insights into the types of DNA lesions that are most important in causing aging in different tissues by allowing impairment of specific DNA repair pathways in defined cell populations. For example, tissue-specific deletion of DNA damage-response/checkpoint genes in DNA repair mutants showing evidence of premature aging might allow the direct effects of these DNA lesions to be distinguished from the cellular responses to them. Further insights into the basic biology of DNA repair proteins, particularly regarding the exact nature of the DNA lesions that these repair systems respond to and their roles in checkpoints, may also shed more light on the role of various DNA lesions in contributing to aging. Eventually, the importance of DNA damage in aging might be directly tested via the generation of experimental organisms with enhanced efficiency of DNA maintenance, which would be predicted to show retarded aging. Although the plethora of different repair systems makes this a challenging, if not impossible, undertaking, the existence of Sir2, a regulator of genome stability and aging in yeast, offers encouragement for those seeking similar global regulators of genome maintenance and potentially DNA repair in mammals.

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Acknowledgments The authors would like to thank members of the Alt lab, as well as Roderick Bronson, Ronny Drapkin, Toren Finkel, Lenny Guarente, Marcia Haigis, F. Bradley Johnson, Michael Lieber, Kevin Mills, Ralph Scully, Peter Sorger, Tony Wynshaw-Boris, and especially Ned Sharpless for helpful discussions and comments on the manuscript. F.W.A. is an Investigator of the Howard Hughes Medical Institute. This work was supported by an Ellison Foundation Senior Scholar Award (to F.W.A), a K08 award from NIA/NIH (to D.B.L.), a Pfizer Postdoctoral Fellowship in Immunology/Rheumatology (to K.F.C.), a Senior Postdoctoral Fellowship from The Leukemia and Lymphoma Society (to R.M.), and a Long-Term Fellowship from the European Molecular Biology Organization (to S.F.). The authors would like to apologize to those whose work was not cited, due to space constraints. We have referenced other articles in this issue and recent in-depth reviews that provide these references.

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