DNA damage and ageing: new-age ideas for an age-old problem

George A. Garinis1,3, Gijsbertus T.J. van der Horst1, Jan Vijg2 & Jan H.J. Hoeijmakers1

Abstract
Loss of genome maintenance may causally contribute to ageing, as exemplified by
the premature appearance of multiple symptoms of ageing in a growing family of
human syndromes and in mice with genetic defects in genome maintenance
pathways. Recent evidence revealed a similarity between such prematurely ageing
mutants and long-lived mice harbouring mutations in growth signalling pathways.
At first sight this seems paradoxical as they represent both extremes of ageing yet
show a similar 'survival' response that is capable of delaying age-related pathology
and extending lifespan. Understanding the mechanistic basis of this response and
its connection with genome maintenance would open exciting possibilities for
counteracting cancer or age-related diseases, and for promoting longevity.

The contribution of endogenous sources of DNA damage

to the multiple mutations in cancer
Aimee L Jackson,
Lawrence A Loeb,
Department of Pathology, University of Washington, Seattle, Washington, WA 98195, USA
Received 1 August 2000, Revised 1 November 2000, Accepted 1 December 2000, Available
online 22 May 2001

There is increasing evidence that most human cancers contain multiple mutations. By the
time a tumor is clinically detectable it may have accumulated tens of thousands of mutations.
In normal cells, mutations are rare events occurring at a rate of 1010mutations per nucleotide
per cell per generation. We have argued that the mutation rates exhibited by normal human
cells are insufficient to account for the large number of mutations found in human cancers,
and therefore, that an early event in tumorigenesis is the development of a mutator
phenotype. In normal cells, spontaneous and induced DNA damage is balanced by multiple
pathways for DNA repair, and most DNA damage is repaired without error. However, in tumor
cells this balance may be shifted such that damage overwhelms the repair capacity, resulting
in the accumulation of multiple mutations. Our hypothesis is that multiple random mutations
occur during carcinogenesis. The sequential mutations that are observed in some human
tumors result from selective events required for tumor progression. We consider the
possibility that endogenous sources of DNA damage, in particular oxidative DNA damage,
may contribute to genomic instability and to a mutator phenotype in some tumors.
Endogenous and environmental sources of reactive oxygen species (ROS) are abundant. In
tumor cells, antioxidant or DNA repair capacity may be insufficient to compensate for the
production of ROS, and these endogenous ROS may be capable of damaging DNA and
inducing mutations in critical DNA stability genes. The possibility that oxidative DNA damage
could be a significant source of the genomic instability characteristic of human cancers is
exciting, because it may be feasible to modulate the extent of oxidative damage through
antioxidant therapy. The use of antioxidants to reduce the extent of molecular damage by
ROS could delay the progression of cancer.
DNA Damage & Repair: Mechanisms for Maintaining DNA Integrity
By: Suzanne Clancy, Ph.D. 2008 Nature Education

Citation: Clancy, S. (2008) DNA damage & repair: mechanisms for maintaining DNA integrity. Nature
Education 1(1):103

Because DNA is the repository of genetic information in each living cell, its integrity and stability are essential to life.
DNA, however, is not inert; rather, it is a chemical entity subject to assault from the environment, and any resulting
damage, if not repaired, will lead to mutation and possibly disease. Perhaps the best-known example of the link
between environmental-induced DNA damage and disease is that of skin cancer, which can be caused by excessive
exposure to UV radiation in the form of sunlight (and, to a lesser degree, tanning beds). Another example is the damage
caused by tobacco smoke, which can lead to mutations in lung cells and subsequent cancer of the lung. Beyond
environmental agents, DNA is also subject to oxidative damage from byproducts of metabolism, such as free radicals. In
fact, it has been estimated that an individual cell can suffer up to one million DNA changes per day (Lodish et al., 2005).
In addition to genetic insults caused by the environment, the very process of DNA replication during cell division is
prone to error. The rate at which DNA polymerase adds incorrect nucleotides during DNA replication is a major factor in
determining the spontaneous mutation rate in an organism. While a "proofreading" enzyme normally recognizes and
corrects many of these errors, some mutations survive this process. Estimates of the frequency at which human DNA
undergoes lasting, uncorrected errors range from 1 x 10-4 to 1 x 10-6mutations per gamete for a given gene. A rate of 1 x
10-6 means that a scientist would expect to find one mutation at a specific locus per one milliongametes. Mutation rates
in other organisms are often much lower (Table 1).
One way scientists are able to estimate mutation rates is by considering the rate of new dominant mutations found at
different loci. For example, by examining the number of individuals in a given population who were diagnosed
with neurofibromatosis (NF1, a disease caused by a spontaneousor noninheriteddominant mutation), scientists
determined that the spontaneous mutation rate of the gene responsible for this disease averaged 1 x 10-4mutations per
gamete (Crowe et al., 1956). Other researchers have found that the mutation rates of other genes, like that for
Huntington's disease, are significantly lower than the rate for NF1. The fact that investigators have reported different
mutation rates for different genes suggests that certain loci are more prone to damage or error than others.

Table 1
DNA Repair Mechanisms and Human Disease

DNA repair processes exist in both prokaryotic and eukaryotic organisms, and many of the proteins involved have been
highly conserved throughout evolution. In fact, cells have evolved a number of mechanisms to detect and repair the
various types of damage that can occur to DNA, no matter whether this damage is caused by the environment or by
errors in replication. Because DNA is a molecule that plays an active and critical role in cell division, control of DNA
repair is closely tied to regulation of thecell cycle. (Recall that cells transit through a cycle involving the G1, S, G2, and M
phases, with DNA replication occurring in the S phase and mitosis in the M phase.) During the cell
cycle, checkpoint mechanisms ensure that a cell's DNA is intact before permitting DNA replication and cell division to
occur. Failures in these checkpoints can lead to an accumulation of damage, which in turn leads to mutations.

Defects in DNA repair underlie a number of human genetic diseases that affect a wide variety of body systems but
share a constellation of common traits, most notably a predisposition to cancer (Table 2). These disorders include
ataxia-telangiectasia (AT), a degenerative motor condition caused by failure to repair oxidative damage in the
cerebellum, and xeroderma pigmentosum (XP), a condition characterized by sensitivity to sunlight and linked to a defect
in an important ultraviolet (UV) damage repair pathway. In addition, a number of genes that have been implicated in
cancer, such as the RAD group, have also been determined to encode proteins critical for DNA damage repair.

Figure 1 : Formation of the most toxic and mutagenic DNA lesion, cyclobutane-pyrimidine dimers by UV
radiation.
Dimers can form between two adjacent pyrimidines. Shown here is (A) thymine-thymine cyclobutane-pyrimidine dimer,
and (B) thymine-cytosine dimer and their photoreactivation by the enzyme photolyase in the presence of light.
Table 2 : Genetic diseases associated with defects in DNA repair systems.
Genetic diseases associated with defects in DNA repair systems.

UV Damage, Nucleotide Excision Repair, and Photoreactivation

As previously mentioned, one important DNA damage response (DDR) is triggered by exposure to UV light. Of the three
categories of solar UV radiation, only UV-A and UV-B are able to penetrate Earth's atmosphere. Thus, these two types
of UV radiation are of greatest concern to humans, especially as continuing depletion of the ozone layercauses higher
levels of this radiation to reach the planet's surface.

UV radiation causes two classes of DNA lesions: cyclobutane pyrimidine dimers (CPDs, Figure 1) and 6-4
photoproducts (6-4 PPs, Figure 2). Both of these lesions distort DNA's structure, introducing bends or kinks and thereby
impeding transcription and replication. Relatively flexible areas of the DNA double helix are most susceptible to
damage. In fact, one "hot spot" for UV-induced damage is found within a commonly mutated oncogene, the p53 gene.

CPDs and 6-4 PPs are both repaired through a process known as nucleotide excision repair (NER). In eukaryotes, this
complex process relies on the products of approximately 30 genes. Defects in some of these genes have been shown
to cause the human disease XP, as well as other conditions that share a risk of skin cancer that is elevated about a
thousandfold over normal. More specifically, eukaryotic NER is carried out by at least 18 protein complexes via four
discrete steps (Figure 3): detection of damage; excision of the section of DNA that includes and surrounds the error;
filling in of the resulting gap by DNA polymerase; and sealing of the nick between the newly synthesized and older DNA
(Figure 4). In bacteria (which are prokaryotes), however, the process of NER is completed by only three proteins,
named UvrA, UvrB, and UvrC.

Bacteria and several other organisms also possess another mechanism to repair UV damage called photoreactivation.
This method is often referred to as "light repair," because it is dependent on the presence of light energy. (In
comparison, NER and most other repair mechanisms are frequently referred to as "dark repair," as they do not require
light as an energy source.) During photoreactivation, an enzyme called photolyase binds pyrimidine dimerlesions; in
addition, a second molecule known as chromophore converts light energy into the chemical energy required to directly
revert the affected area of DNA to its undamaged form. Photolyases are found in numerous organisms, including fungi,
plants, invertebrates such as fruit flies, and vertebrates including frogs. They do not appear to exist in humans, however
(Sinha & Hader, 2002).
Figure 2 : UV radiation can distort DNA, impeding transcription and replication.
A region of DNA is shown before (A) and after (B) its structure is distorted by UV radiation. The distortion in panel B is
due to a convex bend, or kink, in one strand (C).

Figure 3 : The nucleotide-excision repair mechanism in eukaryotes.

When damage causes a distortion in DNA structure (1), an enzyme complex (purple oval) recognizes the distortion (2).
The two strands of the DNA are separated, and single-strand-binding proteins (orange spheres) stabilize the single
strands (3). An enzyme (light blue globular structure) cleaves the distorted strand on both sides of the damage (4). The
damaged part is removed (5), and the gap is filled in by DNA polymerase (blue mitt-shaped structure) and sealed by
DNA ligase (orange mitt-shaped structure) (6).

Additional DNA Repair mechanisms

NER and photoreactivation are not the only methods of DNA repair. For instance, base excision repair (BER) is the
predominant mechanism that handles the spontaneous DNA damage caused by free radicals and other
reactive speciesgenerated by metabolism. Bases can become oxidized, alkylated, or hydrolyzed through interactions
with these agents. For example, methyl (CH3) chemical groups are frequently added to guanine to form 7-
methylguanine; alternatively,purine groups may be lost. All such changes result in abnormal bases that must be
removed and replaced. Thus, enzymes known as DNA glycosylases remove damaged bases by literally cutting them
out of the DNA strand throughcleavage of the covalent bonds between the bases and the sugar-phosphate
backbone. The resulting gap is then filled by a specialized repair polymerase and sealed by ligase. Many such enzymes
are found in cells, and each is specific to certain types of base alterations.

Yet another form of DNA damage is double-strand breaks, which are caused by ionizing radiation, including gamma rays
and X-rays. These breaks are highly deleterious. In addition to interfering with transcription or replication, they can lead
to chromosomal rearrangements, in which pieces of one chromosome become attached to another chromosome.
Genes are disrupted in this process, leading to hybrid proteins or inappropriate activation of genes. A number of cancers
are associated with such rearrangements. Double-strand breaks are repaired through one of two mechanisms:
nonhomologous end joining (NHEJ) or homologous recombination repair (HRR). In NHEJ, an enzyme calledDNA
ligase IV uses overhanging pieces of DNA adjacent to the break to join and fill in the ends. Additional errors can be
introduced during this process, which is the case if a cell has not completely replicated its DNA in preparation for
division. In contrast, during HRR, the homologous chromosome itself is used as a template for repair.
Mutations in an organism's DNA are a part of life. Our genetic code is exposed to a variety of insults that threaten its
integrity. But, a rigorous system of checks and balances is in place through the DNA repair machinery. The errors that
slip through the cracks may sometimes be associated with disease, but they are also a source of variation that is acted
upon by longer-term processes, such as evolution and natural selection.

Figure 4 : Schematic representation of the nucleotide excision repair pathway.

References and Recommended Reading

Branze, D., & Foiani, M. Regulation of DNA repair throughout the cell cycle. Nature Reviews Molecular Cell Biology 9,
297308 (2008) doi:10.1038/nrm2351.pdf (link to article)

Crowe, F. W., et al. A Clinical, Pathological, and Genetic Study of Multiple Neurofibromatosis (Springfield, Illinois,
Charles C. Thomas, 1956)

Lodish, H., et al. Molecular Biology of the Cell, 5th ed. (New York, Freeman, 2004)

Sinha, R. P., & Hder, D. P. UV-induced DNA damage and repair: A review. Photochemical and Photobiological
Sciences 1, 225236 (2002)