First printed in R&D Systems' 2004 Catalog.
The human genome is exposed to potentially deleterious
genotoxic events during every cell division cycle.
The endogenous sources of DNA damage result from cellular metabolism
or routine errors in DNA replication and recombination. In addition,
cellular and organismal exposure to exogenous genotoxic agents such
as ultraviolet (UV) light, oxidative stress, and chemical mutagens,
can lead to a variety of nucleotide modifications and DNA strand breaks.
In order to combat these attacks on the genome, the cell has evolved
a response system that induces cell cycle arrest, allowing sufficient
time for specialized groups of proteins to repair the incurred damage.
The DNA damage response system activates the appropriate DNA repair
pathway, or, in the case of irreparable damage, induces apoptosis.
In the last decade, the characterization of many proteins involved
in sensing and responding to DNA damage has enhanced our understanding
of genotoxic stress responses. Mutations in the genes that encode
DNA damage response proteins can result in a number of genomic instability
syndromes, disorders that often result in a heightened predisposition
to cancer.1 Another hallmark of these disorders is immunodeficiency,
a phenotype caused by an inability to properly repair DNA strand breaks
that occur during development of the immune system. The phenotypes
exhibited by genomic instability syndromes highlight the significance
of proteins that sense, relay, or transduce signals associated with
the genotoxic stress response (Table 1).
|Table 1. Genomic Instability Syndromes
|A-T (Ataxia telangiectasia)
||neurodegeneration, immunodeficiency, premature aging, radiation sensitivity,
|NBS (Nijmegen breakage syndrome)
||microcephaly and mental retardation, immunodeficiency, radiation sensitivity,
|ATLD (A-T-like disorder)
||cerebellar degeneration, radiation sensitivity
||immunodeficiency, premature aging, cancer
||immunodeficiency, premature aging, cancer
|FA (Fanconi anemia)
||congenital abnormalities, bone-marrow failure, cancer
||FANC-A, B, C, D1, D2, E, F, G
|XP (Xeroderma pigmentosa)
||UV light sensitivity, skin aging, skin cancer
||XPA, B, C, D, E, F, G
||nucleotide-excision repair (NER) complex
|CS (Cockayne's syndrome)
||dwarfism, mental retardation, UV light sensitivity
|CSA, CSB, XAB2
Ataxia Telangiectasia and Related Disorders
Ataxia telangiectasia (A-T) is a devastating genetic disease that presents
in the first decade of life. A-T is a multi-symptom disorder characterized
by immunodeficiency, neurodegeneration, premature aging, sensitivity to ionizing
radiation, and susceptibility to certain types of cancer. A-T is ultimately
fatal in the second or third decade of life.2
View Larger Image
|Figure 1. Phosphorylated histone H2AX (gamma-H2AX) nucleates the formation of genotoxic stress response complexes. The phosphorylation of gamma-H2AX, a very early event in genotoxic stress responses, requires ATM or ATM-related protein kinase activity. gamma-H2AX marks the damaged DNA and recruits BRCT-containing proteins, such as 53BP1, MDC1, BRCA1, and Nbs1. ATM or ATM-related kinases can phosphorylate many of these proteins and other downstream targets to relay signals for cell cycle checkpoint activation, DNA repair, or apoptosis. While details regarding protein-protein interactions are only beginning to be elucidated, it is known that 53BP1 recruitment is important for coupling ATM to p53 and SMC1, but not Chk2 activation.
The protein product of the gene mutated in A-T, A-T-mutated (ATM),
is a large protein kinase involved in cell cycle checkpoint and genotoxic
stress responses.3, 4, 5 ATM is a member of the phosphatidylinositol
3-kinase-related kinase (PIKK) family. All PIKKs have a highly conserved
catalytic domain that bears similarity to the catalytic domain of
the lipid kinase phosphatidylinositol 3-kinase (PI 3-K). Despite this
similarity, all of the PIKK family members exhibit protein rather
than lipid kinase activity. ATM and related protein kinases participate
in one of the earliest events that occurs in response to DNA insult
by phosphorylating the C-terminal tail of the core histone H2AX protein
[gamma-H2AX (phosphorylated form); Figure 1].6, 7, 8
gamma-H2AX marks the site of damage and nucleates
the formation of damage response and repair complexes.6, 8 BRCA1
C-terminal (BRCT) domain-containing proteins are recruited to gamma-H2AX
creating a protein scaffold for further assembly of signaling complexes
that include molecules such as p53-binding protein 1 (53BP1),9, 10, 11, 12
mediator of DNA damage checkpoint protein 1 (MDC1),13, 14, 15 and
the BRCA1 tumor suppressor protein.9 Additionally, the BCRT
domain-containing protein Nbs1, the Rad50 and Rad51 DNA repair factors,
and others are recruited to the damage site. For many of these proteins,
specific details regarding direct protein-protein interactions and
order of complex assembly are only beginning to be elucidated. For
example, a recent study suggests that Nbs-1 can directly bind to gamma-H2AX.16
Furthermore, many of the these proteins are substrates for ATM and
related protein kinases, underscoring the critical role of this protein
kinase in genotoxic stress responses.3, 4, 5
While the mutation of both ATM alleles, which results in
A-T, occurs at low frequency (approximately 1 in 40,000 births), it
is estimated that heterozygous carriers constitute 1-2% of the population.3
Carriers may exhibit intermediate sensitivity to radiation and predisposition
to cancer, and thus knowledge of ATM protein function may have broad
implications for public health.3 For instance, if a radiation-sensitive
carrier is diagnosed with cancer, awareness of the ATM mutation
may cause the physician and patient to consider treatment options
other than radiation therapy.
Patients with Nijmegen breakage syndrome (NBS) are immunodeficient
and exhibit increased cancer incidence, but unlike A-T, are not ataxic.17
NBS does affect the nervous system however, as these patients present
with microcephaly and mental retardation.17 The gene mutated
in NBS, Nbs1, encodes a protein that is a downstream substrate
of the ATM protein kinase. Nbs1 is part of a DNA double strand break-binding
complex that also includes the Mre11 and Rad50 gene
nucleating the formation of the Nbs1/Mre11/Rad50 trimer. The Mre11
gene is mutated in another related syndrome, A-T-like disorder (ATLD).19
Patients with ATLD exhibit chromosomal instability, ionizing radiation
sensitivity, and cerebellar degeneration, although they are not immunodeficient
and show no increased incidence of cancer.
Interestingly, while the clinical phenotypes of these diseases are
variable, cultured cells derived from patients exhibit very similar
characteristics. A-T, NBS, and ATLD cells are all sensitive to ionizing
radiation, exhibit chromosomal instability, and possess multiple cell
cycle checkpoint defects.3 One common feature of all three
cell types is radioresistant DNA synthesis (RDS), or the inability
of the cell to suppress DNA synthesis following irradiation.3
When a normal cell is exposed to genotoxic stress, DNA synthesis stops
until the damage is repaired and synthesis can resume. The presence
of RDS implies a defect in S phase, the DNA synthesis checkpoint of
the cell cycle, whereby the cell continues to synthesize damaged DNA
and has the potential to pass on unrepaired errors to daughter cells.
The direct relationships between the mutated genes encoding the
proteins associated with A-T, NBS, and ATLD underscore their participation
in common signaling pathways in response to assaults on the genome.
However, differences in the clinical presentation of these disorders
also suggest that their functions are distinct. There are differences
in the phenotypes of the related mouse models as well. ATM-/-
mice are viable and exhibit many features of A-T, with the noted exception
of neurodegenerative effects. In contrast, Nbs1-/-, Mre11-/-,
and Rad50-/- mice are not viable and expire at various
times during embryonic development.17, 20 However, mice bearing
a truncation in the Nbs1 gene that mimics a mutation found
in humans are viable and exhibit some similarities to NBS patients,
albeit with fewer malignancies.17 Rad50 mutations are
not documented in humans, which may indicate a crucial function for
RecQ DNA Helicase-associated Syndromes
The human RecQ helicase family has 5 members. These proteins bear a high degree
of similarity to the helicase domain of the E. coli RecQ helicase, an
enzyme involved in DNA recombination and repair.21 Three of the
human RecQ proteins are defined as tumor suppressor genes. This is evidenced
by their mutation in three distinct genomic instability syndromes that share
a predisposition to cancer. BLM, WRN, and RTS, the genes
mutated in Bloom's syndrome, Werner's syndrome, and Rothmund-Thompson syndrome,
respectively, encode proteins that contain helicase activities stimulated
by single-stranded DNA binding proteins.22, 23, 24 These helicases play
an important role in regulating recombination, a fact that translates into
elevated levels of recombination, or a "hyperrecombination" phenotype, in
eukaryotic cells deficient in one of these proteins.21 Hyperrecombination
can lead to large deletions that may have an impact on tumor formation.25
All three enzymes have unique substrate specificities for particular types
of DNA, and exhibit very little sequence similarity outside of the helicase
domain. This variation in substrate specificity is consistent with syndrome-specific
clinical features. For instance, Bloom's syndrome patients are susceptible
to a wide range of epithelial and leukemic cancers,26 while Werner's
syndrome leads to a predominance of thyroid cancer, melanoma, and various
sarcomas,21, 27 and Rothmund-Thompson syndrome patients exhibit
an increased prevalence of skin cancer and osteosarcoma.21, 28
Of the three RecQ helicases described above, the functions and interactions
of the WRN and BLM proteins are the best defined. WRN and BLM are
both substrates of the ATM protein kinase and defects in either enzyme
result in the loss of p53-mediated apoptosis.21, 29, 30 One unique
feature of cells from Werner's patients is accelerated senescence,
implying impaired telomere function. Indeed, the WRN protein colocalizes
with telomere-binding proteins, and demonstrates substrate specificity
for telomeric DNA.21 Consistent with the WRN cellular phenotype,
patients with Werner's syndrome exhibit signs of premature aging.21
Fanconi anemia (FA) is characterized by congenital abnormalities
in multiple organ systems, pancytopenia from bone-marrow failure,
chromosomal instability, and increased cancer susceptibility.31, 32 One defining feature of FA is chromosomal sensitivity to DNA crosslinking
agents such as mitomycin C and diepoxybutane.31, 32 Chromosomal
breakage induced by these compounds has been used as a diagnostic
test for FA.32
In contrast to the diseases resulting from A-T-related and RecQ
helicase mutations, relatively little is known regarding the function
of the proteins mutated in FA. Patients with FA are categorized into
8 different complementation groups, and 7 different FA genes including,
FANC-A, FANC-C, FANC-D1, FANC-D2, FANC-E,
FANC-F, and FANC-G have been cloned to date.32
These genes are not clustered on a single chromosome, but are distributed
widely throughout the genome. Of the FA proteins, FANC-A, FANC-C,
FANC-E, FANC-F, and FANC-G contain no obvious catalytic domains and
exist in a nuclear complex (A/C/E/F/G) that possesses no enzymatic
activity.32 However, it has been shown that the A/C/E/F/G complex
mediates the monoubiquitination of FANC-D2 in response to DNA damage.
This post-translational modification results in translocation of FANC-D2
to nuclear foci where DNA-repair complexes including the proteins
BRCA1, BRCA2, and Rad51 are localized.32 It is interesting
to note that the FANC-D1 protein was recently identified as being
identical to the BRCA2 protein, and that patients with BRCA2/FANC-D1
mutations share clinical features with FA.33
FANC-A, FANC-C, FANC-D2, and FANC-G genes
have been targeted in mice, and the resulting phenotype is not as
severe as that found in human FA.32, 34, 35, 36 These mice are sensitive
to DNA crosslinking agents and ionizing radiation, but do not exhibit
anemia. Furthermore, only FANC-D2-deficient mice show an increased
cancer risk, exhibiting a high incidence of epithelial tumors.
The nucleotide-excision repair (NER) process employs an array of more than
30 proteins that detect DNA damage, excise the damaged strand, and synthesize
new DNA using the complementary strand as a template.37, 38 Types
of DNA lesions repaired by NER include cyclobutane pyrimidine dimers and 6-4
photo products resulting from UV light exposure.38 Two autosomal
recessive disorders, Xeroderma pigmentosum (XP) and Cockayne's syndrome (CS),
occur as a result of mutations in NER genes. XP results from defects in one
of seven genes (XPA through XPG), while CS results from mutations
in the genes CSA or CSB.3, 38 Patients with either
disorder are extremely sensitive to UV light while those with XP, specifically,
exhibit a predisposition to cancer.3, 38 Mutations in the XPB
and XPD are also associated with Tricothiodystrophy (TTD), a disorder
in which patients present with mental retardation and UV light sensitivity,
but no increased incidence of cancer.3, 38
These groups of genomic instability disorders have united physicians
treating these patients with researchers interested in the genotoxic
stress response, cell cycle, and DNA repair. As current treatment
options for many of these diseases are limited, a more thorough understanding
of defects at the molecular level will hopefully lead to new drug
targets and ultimately therapies and treatments for these patients.
New insights into genotoxic stress pathways will likely yield additional
treatment options for cancer patients as well.
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- Yu, C.-E. et al. (1996) Science 272:258.
- Kitao, S. et al. (1998) Genomics 54:443.
- Tachibana, A. et al. (1996) Mol. Carcinog. 17:41.
- German, J. et al. (1997) Cancer Genet. Cytogenet. 93:100.
- Goto, M. et al. (1996) Cancer Epidemiol. Biomarkers Prev. 5:239.
- Vennos, E.M. et al. (1992) J. Am. Acad. Dermatol. 27:750.
- Pichierri, P. et al. (2003) Oncogene 22:1491.
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- Ahmad, S.I. et al. (2002) BioEssays 24:439.
- D'Andrea, A.D. & M. Grompe (2003) Nat. Rev. Cancer 3:23.
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