The Importance Of Dna Doublestrand Break Repair

Double-strand breaks (DSBs) in DNA are highly toxic lesions that can be created through a variety of mechanisms, including effects of reactive oxygen species. Double-strand breaks are repaired by either of two mechanistically distinct DNA repair pathways, homologous recombination (HR) or nonhomologous end-joining (NHEJ) (van Gent et al., 2001). A key factor of DSB repair by NHEJ is the DNA-end-binding Ku70/Ku80 heterodimer. Mice harboring a null mutation in the Ku80 gene have a significantly shorter life span and display a range of premature aging phenotypes (Vogel et al., 1999). For this mouse model the age-related phenotypes have been compared to those of their littermate controls in a side-by-side study in an identical environment (same air, same food, same bedding, same cage). Figure 49.1 shows the survival curve of the Ku80 mutant mice, indicating a significantly shorter life span than their littermate controls.

One of the most prominent aging-related phenotypes occurring early in the mutants is lordokyphosis, the lateral curvature of the spine that is also present in the p53 mutants (see Figure 49.2). Lordokyphosis in these mice is likely due to osteoporosis because histology showed the older Ku80-mutant and control bones to exhibit osteopenia (thinning of the bone and reduced trabeculae). Figure 49.3 shows growth plate closure, a well-known age-related phenotype that is a part of maturation, not senescence, in humans. Mice are different

Importance Span Control

Figure 49.1 Life span and mortality of Ku80-mutant and control mice (Vogel et al., 1999). The survival curve begins after weaning (three weeks), because ku80—/— pups are less able to compete than their bigger littermate controls for mother's milk, and often die within the first two weeks (Nussenzweig et al., 1996). Symbols are shown at the points of 100%, 50%, and 0% survival. Number of mice observed: 47 control mice represented by a filled box and 89 ku80—/— mice represented by an open circle.

weeks

Figure 49.1 Life span and mortality of Ku80-mutant and control mice (Vogel et al., 1999). The survival curve begins after weaning (three weeks), because ku80—/— pups are less able to compete than their bigger littermate controls for mother's milk, and often die within the first two weeks (Nussenzweig et al., 1996). Symbols are shown at the points of 100%, 50%, and 0% survival. Number of mice observed: 47 control mice represented by a filled box and 89 ku80—/— mice represented by an open circle.

Figure 49.2 Lordokyphosis in control (+/+ and +/—) and ku80—/— (—/—) mice (Vogel et al., 1999). A. Mice at 2.5 wk. No kyphosis. B. Mice at 31 wk. Kyphosis in only ku80—/— mouse. C. Mice at 75-79 wk. Kyphosis in only ku80—/— mouse. D. Control mice at 120 weeks (ku80—/— mice do not live this long). Kyphosis observed.

Figure 49.2 Lordokyphosis in control (+/+ and +/—) and ku80—/— (—/—) mice (Vogel et al., 1999). A. Mice at 2.5 wk. No kyphosis. B. Mice at 31 wk. Kyphosis in only ku80—/— mouse. C. Mice at 75-79 wk. Kyphosis in only ku80—/— mouse. D. Control mice at 120 weeks (ku80—/— mice do not live this long). Kyphosis observed.

Figure 49.3 Growth plate closure (Vogel et al., 1999). Growth plates look the same for both cohorts between 1 and 15 weeks. However, by 20-45 weeks, compared to controls, the number of chondrocytes is reduced, and the columnar organization of chondrocytes is lost for the ku80—/— epiphysis. This same phenotype is observed for control mice by 70 weeks. Section of epiphysis from control (A, C, E) and ku80—/— (B, D) mice. (A, B), 1-15 weeks (shown are one-week old growth plates). (C, D) 20-45 weeks (shown are 22-week-old growth plates). (E) Greater than 70 weeks of age. Only the control is shown because very few Ku80-mutant mice survive to this age.

Figure 49.3 Growth plate closure (Vogel et al., 1999). Growth plates look the same for both cohorts between 1 and 15 weeks. However, by 20-45 weeks, compared to controls, the number of chondrocytes is reduced, and the columnar organization of chondrocytes is lost for the ku80—/— epiphysis. This same phenotype is observed for control mice by 70 weeks. Section of epiphysis from control (A, C, E) and ku80—/— (B, D) mice. (A, B), 1-15 weeks (shown are one-week old growth plates). (C, D) 20-45 weeks (shown are 22-week-old growth plates). (E) Greater than 70 weeks of age. Only the control is shown because very few Ku80-mutant mice survive to this age.

from humans in that growth plates do not close until well after maturation. Here, growth plates close much earlier in Ku80-mutant mice than in control mice. Figure 49.4 illustrates skin atrophy, a well-described age-related phenotype in both mice and humans. Again, skin atrophy was observed earlier in Ku80-mutant mice than in their littermate controls. In addition, various other aging-related phenotypes were observed in Ku80-mutant mice, well before they appeared in their littermate controls (forms of liver degeneration, reactive immune responses). Even though these phenotypes occur earlier in Ku80-mutant mice than in the controls, they all occur at about the same point in their biological life spans (the latter half of their survival curve). Despite these similarities, there are differences; most obvious is the difference in cancer incidence (cancer incidence is much lower in Ku80-mutant mice than in their littermate controls).

The most straightforward explanation for the accelerated aging phenotypes in Ku80 null mice is increased genomic instability resulting from erroneous or inefficient repair of DNA double-strand breaks in the absence of NHEJ. Such increased genomic instability would trigger apoptosis and interfere with normal cell growth and tissue regeneration. Whereas increased genomic instability normally would be expected to promote tumor formation, the increased rate of apoptosis in the presence of an intact p53 checkpoint is likely to restrain tumor development. This scenario is supported by actual observations. Ku80 mutant mouse cells display growth impairment, increased susceptibility to apoptosis, and a marked increase in chromosomal aberrations, including breaks, translocations, and aneuploidy (Difilippantonio et al, 2000). Results from our laboratories indicate increased genomic instability at a lacZ reporter locus in liver, and, especially, in spleen, already at five months of age. In liver of 10-month-old mice, increased numbers of TUNEL or caspase 3 positive cells were observed, indicating a higher rate of spontaneous apoptosis (Y. Suh and P. Hasty, unpublished results).

Hence, though the inability of the Ku80 mutant mouse to repair DNA damage leads to excessive genome rearrangements, the resulting increase in cell death or dysfunction becomes manifest as impaired proliferation and regeneration, resulting in diminished cancer and accelerated age-related organ and tissue degeneration. This situation may be very similar for two other mouse models with defects in double-strand break repair: mice with defects in DNA-PKCS, the catalytic subunit of the Ku70/Ku80 complex, and mice harboring a hypo-morphic mutation in the BRCA1 gene, a major player in HR. In DNA-PKCS null mice the situation resembles the Ku80 null model: the absence of intact NHEJ promotes genomic instability leading to impaired tissue growth and regeneration (Espejel et al., 2004). Complete loss of BRCA1 is embryonically lethal, and the same is true for the homozygous BRCA1 hypomorph, lacking exon 11. However, the homozygous hypomorph can be rescued completely in a p53 heterozygous background. In a p53-homozygous mutant background, these mice exhibit a high incidence of cancer. In the p53 heterozygous background, BRCA1 hypomorphic mice exhibit a long list of premature aging phenotypes and a significant reduction of life span, probably caused by the activity of the remaining p53 allele, which is triggered by genomic instability to prevent normal cell proliferation (Cao et al., 2003).

Thus far, there is no evidence that inactivation of Ku80's partner in NHEJ (i.e. Ku70) is causing accelerated aging (Gu et al., 1997). However, Ku70 null mice have never been studied as an aging cohort over longer periods of time in parallel with their littermate controls. Such studies are now underway and should soon reveal if there really is a difference between the Ku80 and Ku70 mutants. It is possible that each of these two key players in DSB repair has other, tissue-specific functions that confound their role in suppressing aging.

AGING, DNA REPAIR, AND TRANSCRIPTION

Another type of DNA repair defect associated with premature aging involves transcription-related nucleotide excision repair (NER). NER removes a broad range of helix-distorting lesions, from UV-induced DNA damage and numerous chemical adducts to oxidative damage produced by endogenous metabolism (Hoeijmakers, 2001). Within NER two subpathways are recognized, differing in damage recognition but sharing the same repair machinery: global genome NER (GG-NER) for the removal of distorting lesions anywhere in the genome and transcription-coupled NER (TC-NER) for the elimination of distorting DNA damage blocking transcription. Two mouse models with defects in NER have been reported to display premature aging symptoms: the Xpd hypomorph (de Boer et al., 2002) and the Ercc1 knockout or hypomorph (Weeda et al., 1997).

In humans, a heritable mutation in the XPD gene is responsible for the disorder trichothiodystrophy (TTD). TTD shows no predisposition to cancer, but leads to severely impaired physiological and neurological development, including retarded growth, cachexia, sensori-neural hearing loss, retinal degeneration and its hallmark features of brittle hair, nails, and scaly skin (Lehmann, 2003). TTD patients have a strongly reduced life span, and the disease often is considered as a segmental progeroid syndrome. The helicase encoded by the XPD gene is one of the 10 subunits of basal transcription factor IIH (TFIIH), which is required for multiple processes: GG-NER, TC-NER of NER and non-NER lesions, as well as transcription initiation by RNA polymerase I and II.

An Xpd-deficient mouse model was generated by mimicking a human mutation that causes TTD (de Boer et al., 1998). This mutation does not ablate but rather alters the normal activity of Xpd. These mice have impaired transcription and mildly impaired NER.

Figure 49.4 Skin atrophy (Vogel etal., 1999). Section of skin (dorsal region over cranial to mid-thorax) from control (A, C, E) and ku80—/— (B, D) mice. Skin looks the same for both cohorts between 1 and 15 weeks. However, by 40 weeks, compared to control, all subcutaneous elements, including superficial collagen, subcutaneous adipose and skeletal muscle are atrophied for ku80—/— skin. This same phenotype is observed for control mice by 70 weeks.

Figure 49.4 Skin atrophy (Vogel etal., 1999). Section of skin (dorsal region over cranial to mid-thorax) from control (A, C, E) and ku80—/— (B, D) mice. Skin looks the same for both cohorts between 1 and 15 weeks. However, by 40 weeks, compared to control, all subcutaneous elements, including superficial collagen, subcutaneous adipose and skeletal muscle are atrophied for ku80—/— skin. This same phenotype is observed for control mice by 70 weeks.

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