DNA DAMAGE AND REPAIR
prepared by David Mitchell
In: A Handbook of Organic Photochemistry and Photobiology,
William Horspool and Pill-Soon Song, Eds., CRC Press, London
The amount of solar ultraviolet radiation (UVR) reaching the earth's surface
has increased due to chlorofluorocarbon pollution of the upper atmosphere.
The consequences of stratospheric deozonation on the human population are
difficult to predict, but may include increased skin cancer and accelerated
aging; less direct effects may include deterioration of natural ecosystems
and reduction of major food crops (Calkins 1982).
Due to its maximum absorbance at 260 nm, DNA is considered the primary chromophore
of UVR. The absorption spectrum of DNA correlates well with photoproduct formation,
cell killing, mutation induction, and tumorigenesis. Because UVR induces numerous
structural lesions in DNA, sensitive and precise measurements of DNA damage
and repair are essential for understanding the cytotoxic and mutagenic effects
of specific photoproducts on individual cells and complex organisms.
The sun emits energies at wavelengths that range through 11 orders of magnitude,
from 10^(-4) nm to 10^(12) nm (Smith 1977). The vast majority of this energy
is biologically irrelevant; ionizing radiations such as high energy particles,
X-rays and gamma rays are expended by atomic collisions in the upper atmosphere
and long wavelength far infrared, microwaves and radiowaves do not have sufficient
energy to influence biochemical reactions. Photodependent biological processes
derive their energy from the UV and visible portions of the solar spectrum.
UVR is divided into the UVC (240-290 nm), UVB (290-320 nm) and UVA (320-400
nm) regions. Absorption of UVC irradiation by stratospheric ozone greatly
attenuates these wavelengths, resulting in very little light shorter than
300 nm reaching the earth's surface. Hence, although comprising only a negligible
portion of sunlight, UVB is responsible for most of the sun's pathological
UVR is a potent and ubiquitous carcinogen responsible for much of the skin cancer in the human population today (Cleaver and Kraemer 1989). Tumor incidence and mortality correlate with exposure: basal and squamous cell carcinomas are most prevalent on the face and trunk in men and the face and legs in women; carcinomas increase with decreasing latitude; tumor incidence is increased in individuals working in occupations with high exposure, such as ranchers or fisherman; and the protective action of skin pigmentation results in lower cancer rates in dark-skinned populations compared to lighter-skinned peoples. Genetic diseases that greatly increase the risk of sunlight-induced skin cancer exemplify the importance of UVR damage and its repair in humans. In one such disease, xeroderma pigmentosum, a failure in the DNA repair process is associated with a major increase in the rate of squamous and basal cell carcinoma and melanoma.
The biological effects of DNA photodamage depend on the type of lesion induced
and its genomic location, as well as the nature of the target cell and its
developmental state. If the photoproduct is repaired correctly, the DNA is
restored to its original state and, after some delay, the cell proceeds with
its normal activities. If the damage is not repaired, it may interfere with
DNA replication or transcription. Should this occur, cell proliferation will
cease or, if the block is situated in a gene required for an essential metabolic
function, the cell will die. Alternatively, the damage may not block replication.
Bypass of the lesion by a DNA polymerase may produce an incorrect complementary
base (i.e., a mutation). This mutation can have several outcomes: (1) It may
not alter the genetic code and, hence, not effect normal metabolism. (2) It
may produce a truncated or partial RNA transcript and manufacture a dysfunctional
protein. If this protein is essential, then the mutation is lethal. (3) It
may result in activation of an oncogene or inactivation of a tumor suppressor
gene, thereby initiating the carcinogenic process (slide
Photon absorption rapidly (10^(-12) sec) converts a pyrimidine base to an
excited state. Various pathways are then available for resolution of this
unstable electronic configuration (Wang 1976). The major pathway involves
rapid dissipation of the energy of the excited base to the ground state (10^(-9)
sec) by non-radiative transition or by fluorescence, yielding heat or light
in the process. Secondly, the excited base can react with other molecules
to form unstable intermediates (i.e. free radicals) or stable photoproducts.
Finally, there is a low probability that intersystem crossing, a nonradiative
pathway, can transfer a base from the excited singlet state to the excited
triplet state. The lifetime of the triplet state is several orders of magnitude
longer than the excited singlet state (10^(-3) sec), increasing the chance
of photoproduct formation. Cyclobutane dimers form through the excited triplet
state. Other photoproducts, such as the pyrimidine(6-4) pyrimidone photoproduct
[or (6-4) photoproduct] form by some other mechanism.
Dimerizations between adjacent pyrimidine bases are by far the most prevalent
photoreactions resulting from UVC or UVB irradiation of DNA. The relative
induction of these photoproducts depends on wavelength, DNA sequence, and
protein-DNA interactions. The two major photoproducts are the cyclobutyl pyrimidine
dimer and the (6-4) photoproduct (slide 2).
The (6-4) photoproduct undergoes a further UVB-dependent conversion to its
valence photoisomer, the Dewar pyrimidinone (slide
2). In addition to the major photoproducts, rare dimers may also form,
such as the adenine-thymine heterodimer.
In contrast to the direct induction of DNA damage by UVC and UVB light, UVA
produces damage indirectly through highly reactive chemical intermediates.
Similar to ionizing radiation, UVA radiation generates oxygen and hydroxyl
radicals which in turn react with DNA to form monomeric photoproducts, such
as cytosine and thymine photohydrates, as well as strand breaks and DNA-protein
crosslinks. The relationship between the relative occurrence of these photoproducts
and their biological effect depends on the cytotoxic and mutagenic potential
of the individual lesion. Hence, even though a photoproduct may occur at a
low frequency, its structure and location may elicit a potent biological effect.
A variety of analytical procedures have been developed to measure UVR damage
and repair in DNA (Friedberg and Hanawalt). Many types of photoproducts have
been identified and quantified by chromatographic techniques. Since the initial
detection of the cyclobutane dimer by 2-dimensional paper chromatography,
thin-layer chromatography and high performance liquid chromatography have
been adapted to the analysis of this and other types of photodamage. All of
these procedures require DNA that has been hydrolyzed to free bases or nucleotides
by enzymatic digestion or treatment in strong acid at high temperature.
Many types of photodamage can be converted into single-strand breaks in DNA
by enzymatic or biochemical treatment. Frank breaks induced directly in DNA
by UVR and breaks produced at sites of photoproducts can be quantified by
alkaline gradient centrifugation, alkaline elution, or agarose gel electrophoresis.
The sensitivity of these procedures requires maintenance of high molecular
weight DNA through the extraction and analytical procedures. UvrABC exinuclease
(slide 3), a partial excision repair complex
purified from Escherichia coli cleaves DNA on either side of damage
produced by exposure to UVR (slide
4). Cleavage of specific photoproducts has been achieved by using
purified enzymes that combine a glycolsylase that cuts the base from the sugar
leaving an abasic (AP) site, and an apurinic/apyrimidinic endonuclease which
cleaves the phosphodiester backbone on either side of the AP site. Nonenzymatic
cleavage of alkali-labile sites, such as AP sites or Dewar pyrimidinones,
also produces quantifiable strand breaks for analysis of induction and repair.
These techniques have recently been adapted to the analysis of UV photoproduct
induction and repair at the gene and base sequence levels. Photodamage at
the sequence level can be mapped as strand breaks in DNA fragments irradiated
with high fluences of UVC and UVB light and at the gene level using Southern
blot hybridization of DNA fragments separated by agarose gel electrophoresis.
Recently, the distribution of photoproducts at the sequence level in a gene
promoter irradiated in vivo with low UV fluences was analyzed by ligation-mediated
polymerase chain reaction.
Polyclonal and monoclonal antibodies recognize and bind a variety of photoproducts,
including the cyclobutane dimer, (6-4) photoproduct, Dewar pyrimidinone, and
thymine glycol (slide 5). Techniques such
as immunoprecipitation, enzyme-linked immuoassays, radioimmunoassays, quantitative
immunofluorescence, and immuno-electron microspcopy are powerful analytical
tools; each has its own unique attributes and applications. Unlike chromatography,
immunological assays do not require chemical or enzymatic degradation of DNA
before analysis; unlike assays that require strand scission, sensitivity does
not depend on the molecular weight or purity of sample DNA.
The total number of photoproducts remaining in cellular DNA at any time depends
on the amount of UVR absorbed and the amount of damage repaired. Damage tolerance
strategies are complex and vary greatly among organisms. Nearly all organisms
have behaviors or natural features that mitigate exposure of DNA to solar
UVR and reduce the amount of photodamage. In addition to habitat selection,
avoidance responses, and physical morphologies which attenuate the transmission
of UVR to sensitive, internal areas of cells, many organisms have evolved
biochemical mechanisms to protect themselves. UVR absorbing compounds such
as melanin and anthocyanin are produced in human skin and in plants; colorless
UV-absorbing compounds, such as flavenoids in terrestrial plants, mycosporine
amino acids in fungi and mycosporine-like compounds in marine organisms, have
also been identified as possible UV-protective chemicals.
Once DNA damage is present, its removal may proceed in whole or in part by
at least two well-studied mechanisms, photoenzymatic repair (PER)(slide
6) or excision repair (Friedberg 1984). PER involves specific recognition
and binding of a small enzyme called a photolyase to a cyclobutane dimer.
This enzyme catalyzes the direct reversal of the dimer upon absorption of
a photon within the UVA/visible range of light. Photoreactivation occurs widely
throughout the plant and animal kingdoms. Its presence and operation in placental
mammals, particularly in human skin, is a matter of some controversy.
Organisms that display reduced PER, often have a greater capacity for excision
repair. The excision repair mechanism utilized by the cell depends on the
type of damage encountered and the location of the damage in chromatin. The
excision repair pathway begins with the search and recognition of the lesion
as a structural distortion in the DNA helix. DNA at the site of damage is
unwound or otherwise disassociated from nucleosomal proteins to provide accessibility
to repair enzymes. The DNA backbone is then incised at or near the site of
the lesion allowing its excision and concomitant resynthesis of the DNA around
the damaged site. Finally, the single-strand gap remaining after disengagement
of the DNA polymerase complex is ligated.
Base excision repair is initiated by enzymatic recognition of the lesion
and scission of the bond between the damaged base and its associated deoxyribose
sugar, a process called aglycosylation. Examples of n-glycosylases include
endonuclease III from E. coli which repairs photohydrates and endonuclease
V from T4 phage which recognizes cyclobutane dimers. In the latter case, cleavage
of the n-glycosyl bond of the 5' pyrimidine base leaves an abasic site which
is subsequently recognized by an AP endonuclease that cleaves the phosphodiester
bond 3' to the abasic site. The remaining abasic site is digested with a 5'
AP endonuclease or 3'6 5' exonuclease associated with DNA polymerase. The
damaged strand is removed and resynthesized by a DNA polymerase complex and
the remaining gap is ligated.
In contrast to base excision repair, nucleotide excision repair recognizes
a broad spectrum of DNA damage, including UVR-induced photoproducts. This
process has been well studied in E. coli (slide
7) and is thought to be an important excision repair pathway in eukaryotes
as well (slide 8). As with base excision
repair, the nucleotide excision repair process is initiated by enzymes that
recognize and bind the helical distortion created at the damaged site. A repair
complex is assembled and cleaves the DNA a few bases to either side of the
lesion, leaving a gap. This gap is filled by a DNA polymerase and the strand
is ligated to restore the DNA duplex to its original integrity.
The clinical effects of defective DNA repair can include increased risk of skin cancer and accelerated aging, as well as neurological and growth disorders. This phenotypic heterogeneity belies the complexity of the excision repair process and presents a formidable task in defining the functions of the various proteins involved in DNA repair. Recent advances in cloning the genes involved in DNA repair in mammals, yeast and Drosophila melanogaster have greatly increased our understanding of the DNA repair process. In addition to defining the components of an ancient and essential molecular process, future studies in DNA repair may help direct the prevention and clinical treatment of sunlight-induced skin cancer and lessen the impact of stratospheric deozonation on the human population.
1. J. Calkins (Ed.), The Role of Solar Ultraviolet Radiation in Marine Ecosystems, NATO Conference Series IV: Marine Sciences, Vol. 7, Plenum Press, New York, 1982.
2. Smith, K. C. (Ed.), The Science of Photobiology. Plenum Press, New York (1977).
3. Cleaver, J. E. and Kraemer, K. H., Xeroderma pigmentosum, in: The Metabolic Basis of Inherited Disease, Vol. II, 6th Ed. (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.), McGraw-Hill, 1989, pp. 2949-2971.
4. Wang, S. Y. (Ed.), Photochemistry and Photobiology of Nucleic Acids, Vols. I and II. Academic Press, New York, 1976.
5. Friedberg, E. C. and Hanawalt, P. C. (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Vols. 1-3. Marcel Dekker, Inc., New York.
6. Friedberg, E. C., DNA Repair, W.H. Freeman and Co., New York, 1984
last modified on Feb 3, 2009