Lecture 15.

Excision repair
Nuclear DNA is a sensitive target-there are only two copies of the essential genes

Defects in DNA-much more serious than defects in other cellular components, which may
be present in multiple copies and readily replaced by metabolic processes

Defects in DNA can be repaired (other cellular components are mostly recycled), however
DNA repair is not perfect and results in mutation

Difference between DNA damage and mutation (draw) but-both damaged DNA and
mutations accumulate

This resulted in a somatic mutation theory of aging, which says that aging
results from accumulation of somatic mutations, leading to impaired function of organs
and tissues.
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Objections: is the spontaneous mutation rate-enough to explain age-related changes?
E. coli 10-5 – 10-6 per locus per generation, human cells 2x10-7 per generation, typical cell
would not accumulate more than a few mutations in a lifetime

But per unit time mutation rate is almost 1 in a 1000 per locus per 100 years
Human genome is estimated 30,000 genes means 30 mutant genes per cell at old age
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Mutations are needed-they are the ultimate source of genetic variation for evolution
But too many mutations reduce fitness and lead to population extinction

Also for multicellular organisms, somatic cells are unlikely to maintain mutation rates
lower than just necessary to reach the age of reproduction

Sources of DNA damage

Replication errors
Chemical lesions from environmental and endogenous agents (oxygen free radicals)
UV light, ionizing radiation, chemicals

Types of damage
Chemical modification of DNA bases, cross-links, bulky adducts, single-and double-
stranded breaks

The modified bases and other damages that leave one strand intact, are removed by
excision repair pathways
PICT Base excision repair

Deletion of XRCC1 gene – essential for all BER is embryonic lethal

Deletion of any of DNA glycosylases-generally has no obvious phenotype

No human disease with mutation in essential component of BER-perhaps embryonic lethal

PICT Nucleotide excision repair (TCR and global repair)

XPA, XPC recognition

XPB, XPD, TFIIH helicases
XPF (ERCC1), XPG incision

CSB, CSA TCR repair, interact with stalled DNA polymerase

NER mutations are associated with 3 human heritable disorders:

Xeroderma pigmentosum XP
Trichothiodystrophy TTD
Cockayne syndrome CS

PICT-XP

XP-heterogeneous disease can be caused by mutations in different genes from NER pathway
Individuals with XP have >1000 fold increase in UV-induced skin cancer, accelerated aging of
the skin, frequently neurodegeneration.

Aged appearance of skin, most patients die of cancer before adulthood-no other clear symptoms
of accelerated aging

Mice with deletion of XPA gene-develop normally-increased skin cancer in old mice-no data no
pronounced accelerated aging (up to 15 mo)
(mice have limited exposure to UV-the source of NER damage)

TTD and CS-prominent symptoms of accelerated aging

TTD-point mutations in XPD gene. Different mutations can give rise to XP, CS or TTD

XPD-encodes helicase that is part of the TFIIH transcription factor-unwinds DNA for
transcription and also for NER-the phenotype can be caused by either defect in transcription or
defect in repair

PICT-brittle hair-deficiency in sulfur proteins

TTD-neurological and skeletal degeneration, aging in facial features, cachexia, ichthyosis,
brittle hair and nails, physical and mental retardation, “failure to thrive”, dwarfism
Sun light causes pain-child screams in pain when exposed to sun light
But no increase cancer incidence
PICT-mice-
PICT-mice-

Mice with mutation in XPD-also show premature aging phenotypes

Smaller and tinier. Also, brittle hair and skin lesions
Osteoporosis, grey hair
Organs have aged histology, loss of hypodermal fat, accumulation of lipofuscin-oxydized lipid-
linked to oxidative damage, curved spine
They are fertile, but prematurely loose fertility

PICT-reduced ls
PICT-cause of death
PICT-all symptoms get more severe when crossed to XPA mice
Table-cause of death (die of anorectal prolaps-muscle weakness)
PICT-DNA repair
Strange-look calorically restricted-could be the cause of reduced cancer

CS
PICT-Cockayne syndrome
Defect in TCR
Mutations in CSA or CSB proteins

Human patients-aging related phenotypes, cachexia (loss of weight, muscle weakness, loss of
appetite), neuronal degeneration
No increased susceptibility to cancer
The oldest CS patient is in the late 30s. Many CS patients die in infancy.
CS mice-are not as severely affected as humans, but develop neurological abnormalities, tremor,
limb ataxia, inner ear defects, cachexia

Modest increase in cancer incidence, all symptoms get more severe when crossed to XPA mice

Downregulation of insulin/IGF signaling in mice with NER defects

Their transcriptional profile is similar to CR mice, also similar to the mice subjected to oxidative
stress.
The phenotypes like dwarfism may be the result of stress response-activation of stress program,
similar to long-lived dwarf mice. However NER mutants cannot live long due to DNA damage.

Different mutations lead either to cancer or to aging or combination of both, the mechanisms is
not clear.
Possibly, TCR defects cause strong activation of tumor-suppressor pathways, decrease in cancer
but causes aging.
Efficiency of repair in different species:

PICT Unscheduled DNA synthesis

Rodent repairdox

Human fibroblasts remove 50-80% of lesions in 24 h

Mouse fibroblasts remove 10-30%
However, cell survival (colony forming ability) is similar, i.e. mice survive without repair.

Rodents (mice, hamsters) are deficient in repair of pyrimidine dimmers.

P48-XPE gene is methylated (in hamster) or otherwise repressed-nocturnal hairy creatures, no
UV exposure

What happens to excision repair during aging?

The data is complex and unclear

We have a presentation today about some modern studies, new methods

Old studies:

Vijg J, Mullaart E, Lohman PH, Knook DL.

UV-induced unscheduled DNA synthesis in fibroblasts of aging inbred rats.
Mutat Res. 1985 Sep;146(2):197-204.

Wistar (WAG/Rij) rats. Skin fibroblasts were isolated at 2 time points, separated by a 9-month
interval, from rats of various ages. The isolated cells were cultured for 1 passage, irradiated with
ultraviolet light (UV) and analyzed by autoradiography for their capacity to perform UDS. The
results of the two cross-sectional series of determinations were identical: small variations among
individual animals and a slight, but statistically significant age-related decrease in the initial rate but
not in the end level of UV-induced UDS. The small variation among individual inbred rats as
compared with the large variation reported for UDS in human populations suggests that the latter is
largely due to genetic differences. The lack of a more pronounced age-related decrease along with
the small individual variation suggests that the activity of the DNA nucleotide excision repair
pathway is not an important single determinant of individual longevity in inbred rats of the same
strain and sex.

Mullaart E, Roza L, Lohman PH, Vijg J.

The removal of UV-induced pyrimidine dimers from DNA of rat skin cells in vitro and in vivo in relation to aging.
Mech Ageing Dev. 1989 Mar;47(3):253-64.
Removal of UV pyrimidine dimers in rats of different age-used micrococcal endonuclease
that makes single-strand breaks next to dimers
PICT-no difference with age

Human studies
Phil Hanawalt:
Liu SC, Parsons CS, Hanawalt PC.
DNA repair response in human epidermal keratinocytes from donors of different age.
J Invest Dermatol. 1982 Nov;79(5):330-5.

Liu SC, Parsons S, Hanawalt PC.

DNA repair in cultured keratinocytes.
J Invest Dermatol. 1983 Jul;81(1 Suppl):179s-83s.
DNA replication was inhibited by UV radiation to an equal extent in cell cultures from newborns
and adults. After a UV dose of 13 J/m2, the time course of DNA repair was similar for the
respective cultures. Our comparative studies of DNA repair in keratinocytes from infant and aged
donors have revealed no significant age-related differences for repair of UV-induced damage to
DNA. We conclude that no significant age-related differences exist in the rate and extent of the
repair-replication response of human epidermal keratinocytes to UV-radiation damage in DNA.

Decline was observed in smokers-measured UDS

Stierum RH, Hageman GJ, van Herwijnen MH, van der Veer MS, Kleinjans JC.
Age-related negative associations between parameters of cytogenetic damage and ex vivo (+/-)-anti-benzo(a)pyrene
diolepoxide-induced unscheduled DNA synthesis in smoking humans.
Cancer Epidemiol Biomarkers Prev. 1997 Nov;6(11):943-8.