This document discusses how DNA damage occurs through endogenous and exogenous sources, and the importance of DNA repair in maintaining genomic integrity and preventing cancer. It describes various types of DNA damage including base modifications, single-strand breaks, and double-strand breaks that can be caused by reactive oxygen species, radiation, chemicals and replication errors. The document outlines different DNA repair pathways including base excision repair, nucleotide excision repair, mismatch repair, non-homologous end joining and homologous recombination that evolved to fix different types of DNA lesions. Defects in DNA repair genes are associated with increased cancer risks demonstrating the critical role of DNA repair in preventing tumorigenesis.

1. Maintenance of Genomic Integrity
and the Development of Cancer

2. DNA Damage
DNA damage, due to environmental factors and
normal metabolic processes inside the cell, occurs at a
rate of 1,000 to 1,000,000 molecular lesions per cell
per day.

3. Failure to repair DNA lesions may result
in blockages of transcription and
replication, mutagenesis, and/or cellular
cytotoxicity.
In humans, DNA damage has been shown
to be involved in a variety of genetically
inherited disorders, in aging, and in
carcinogenesis.

4. Cell genomes are threatened by errors made
during DNA replication
- The stability of genome is under constant
challenge by a variety of agents and processes:
1. The replication of DNA is subjected to a low but
significant level of error. – incorporation of chemically
different nucleotide precursors
2. The nucleotides within DNA molecules undergo chemical
changes spontaneously.
3. DNA molecules may be attacked by various mutagenic
agents, including endogenous and exogenous agents.

5. Sources of DNA Damage
DNA damage can be subdivided into two main
types:
1. endogenous damage such as attack by reactive
oxygen species produced from normal metabolic
byproducts (spontaneous mutation), especially
the process of oxidative deamination;
• also includes replication errors

6. 2.exogenous damage caused by external agents
such as
• ultraviolet [UV 200-300nm] radiation from the
sun
• other radiation frequencies, including x-rays and
gamma rays
• human-made mutagenic chemicals, especially
aromatic compounds that act as DNA
intercalating agents

7. Cell genomes are under constant attack from
endogenous biochemical processes
- Endogenous biochemical processes may make
greater contributions to genome mutation than
do exogenous mutations.
- DNA molecules are subjected to chemical
damage through the actions of hydrogen and
hydroxy ions that are present at low
concentration (~ 10-7 M) at neutral pH.

8. Mechananisms of damage at molecular level

9. Direct action of ionizing radiation
Ionizing radiation + RH R- + H+
OH
I
R – C = NH
imidol (enol)
O
II
R – C = NH2
amide (ketol)
 
Tautomeric shifts
Bond breaks

10. e-
X ray
 ray P+
O
H
H OH-
H+
Ho
OHo
Indirect action of ionizing radiation

11. The most important types of radiation induced lesions
in DNA
Single-strand breaks
500-1000 per 1 Gy
Double strand breaks
40-50 per 1 Gy
Base damage: 1000-2000 per 1 Gy

12. So thank goodness for
DNA Repair

13. IMPORTANCE OF DNA REPAIR

14. © John Wiley & Sons, Inc.
Types of Mutations
• Changes in chromosome number and structure
• Point mutation--changes at specific nucleotide in a gene
(A,T,C,G)
• Insertion mutations--insert fragment of DNA
• Deletion mutations--delete fragment of DNA

15. Types of Damage
The main types of damage to DNA due to
endogenous cellular processes:
1. oxidation of bases [e.g. 8-oxo-7,8-
dihydroguanine (8-oxoG)] and generation of DNA
strand interruptions from reactive oxygen
species.
2. alkylation of bases (usually methylation), such
as formation of 7-methylguanine, 1-
methyladenine, O6 methylguanine
3. hydrolysis of bases, such as deamination,
depurination and depyrimidination.

16. 4. "bulky adduct formation" (i.e. benzo[a]pyrene
diol epoxide-dG adduct).
5. mismatch of bases, due to errors in DNA
replication, in which the wrong DNA base is
stitched into place in a newly forming DNA
strand, or a DNA base is skipped over or
mistakenly inserted.

17.  Accuracy of the DNA replication
machinery
 Efficiency of the mechanisms for the repair
of damaged DNA
 Degree of exposure to mutagenic agents
in the environment
Factors Influencing the Rate of Spontaneous
Mutations

18. Mutation Frequency
are infrequent
 Bacteria and phage: 10–8 to 10–10 per
nucleotide pair per generation
 Eukaryotes: 10–7 to 10–9 per nucleotide
pair per generation
1/107 to 1/109
Silent mutation: UCU=Ser;
UCA, UCC, UCG = Ser

19.  Ionizing radiation breaks
chromosomes and can cause deletions,
duplications, inversions, and
translocations
Ionizing Radiation Causes Changes in
Chromosome Structure

20.  Single-strand and double-strand
breaks are produced at low frequency
during normal DNA metabolism by
topoisomerases, nucleases, replication
fork "collapse", and repair processes.
 Breaks are also produced by ionizing
radiation.
Strand breaks

21. cyclobutane pyrimidine dimers pyrimidine (6-4) pyrimidinone
(60% T-T, 30% C-T, 10% C-C dimers)
Products of UV irradiation

22. Cells deploy a variety of defenses to
protect DNA molecules from attack
by mutagens
- Physical shield: skin and the melanin pigment
- detoxifying enzymes: superoxide dismutase (SOD)
& catalase
- free-radical scavengers: vitamin C, vitamin E, bilirubin
- glutathione-S-transferases (GSTs)
reacting with electrophilc compounds

23. Melanin pigment shields keratinocyte
nuclei from UV radiation
supranuclear cap
(parasol or sun umbrella)
keratinocyte nucleus

24. Repair enzymes fix DNA that has been
altered by mutagens
- If genotoxic chemicals are not intercepted before
they attack DNA, mammalian cells have a backup
strategy for minimizing the genetic damage caused
by these potential carcinogens.

25. - A cell has two major strategies for detecting and
removing the miscopied nucleotides arising
during DNA replication.
1. Proofreading by DNA polymerases
2. DNA repair by mismatch repair (MMR) enzymes

26. Proofreading by DNA polymerases
δ

29. Mismatch repair (MMR) enzymes detect
mistakes in newly synthesized DNA strand
Two components of the
MMR apparatus, MutS and
MutL, collaborate to
remove mismatched DNA:
- MutS scans the DNA
for mismatches.
- MutL then scans the DNA
for single-strand nicks,
which identify the strand
that has recently been
synthesized.

30. Base-excision repair (BER)
cleave the glycosyl bond
linking the altered base
and the deoxyribose
apurinic/apyrimidinic
endonuclease

32. Nucleotide-excision repair (NER)
PCNA: proliferation-
cell nuclear antigen
RPA: single-strand
DNA-binding protein
NER is accomplished by
a large multiprotein
complex composed of
almost ~20’s subunits.

34. - NER can be divided into 2 subtypes:
transcription-coupled repair (TCR)
global genomic repair (GGR)

35. TCR and GGR

36. Inherited defects in nucleotide-excision repair
(NER) and mismatch repair (MMR) lead to
specific cancer susceptibility
NER defect: Xeroderma pigmentosum (XP)
MMR defect: Hereditary non-polyposis colon cancer
(HNPCC)

37. Double-strand breaks
Double-strand breaks (DSBs), in which both
strands in the double helix are severed, are
particularly hazardous to the cell because they
can lead to genome rearrangements.
Various mechanisms exist to repair DSBs:
1) non-homologous end joining (NHEJ),
2) recombinational repair (also known as template-
assisted repair or homologous recombination
repair)

40. An option: DNA damage checkpoints
 After DNA damage, cell cycle checkpoints are
activated.
 Checkpoint activation pauses the cell cycle and gives
the cell time to repair the damage before continuing
to divide.

42. DNA Damage Response (DDR)

43. In NHEJ
DNA Ligase IV, a
specialized DNA Ligase
that forms a complex
with the cofactor
XRCC4, directly joins the
two ends.

44. Recombinational repair requires the presence of an
identical or nearly identical sequence to be used as a
template for repair of the break.
The enzymatic machinery responsible for this repair
process is nearly identical to the machinery responsible
for chromosomal crossover during meiosis.
This pathway allows a damaged chromosome to be
repaired using a sister chromatid (available in G2 after
DNA replication) or a homologous chromosome as a
template.
Recombinational Repair (HR)

46. Clear association of DNA repair and cancer
Inherited mutations that affect DNA repair genes are
strongly associated with high cancer risks in humans.
Hereditary nonpolyposis colorectal cancer (HNPCC) is
strongly associated with specific mutations in the DNA
mismatch repair pathway.
BRCA1 and BRCA2, two famous mutations conferring a hugely
increased risk of breast cancer on carriers, are both
associated with a large number of DNA repair pathways,
especially NHEJ and homologous recombination.

47. BRCA1, BRCA2
Homologous recombination repair (HRR) of double-strand breaks and daughter strand gaps Breast, ovarian
ATM
NHEJ or homology-directed DSBR (HDR) Leukemia, lymphoma and breast cancer
NBS
NHEJ Lymphoid malignancies
MRE11
HRR Breast
BLM
HRR Leukemia, lymphoma, colon, breast, skin, auditory canal, tongue, esophagus, stomach, tonsil, larynx, lung and uterus.
WRN
HRR and NHEJ as well as long-patch BER soft tissue sarcomas have a particularly high incidence in WS, but also colorectal, skin,
thyroid, and pancreatic cancers
RECQ4 (RECQL4)
causing Rothmund-Thomson syndrome (RTS), RAPADILINO syndrome or Baller Gerold syndrome likely HRR
cutaneous carcinomas, including basal cell carcinoma, squamous cell carcinoma, and Bowen's disease (intraepidermal
carcinoma characterized by the development of pink or brown papules and sarcomas are the second most frequently reported
malignancy, at a frequency of 9% of RTS cases
FANCA, FANCB, FANCC, FANCDl, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN
HRR and TLS leukemia, liver tumors, solid tumors in many locations
XPC, XPE(DDB2) XPA, XPB, XPD, XPF, XPG
NER(GGR type) skin cancer (melanoma and non-melanoma)
XPV(POLH)
TLS skin cancer (melanoma and non-melanoma)
hMSH2, hMSH6, hMLH1, hPMS2
MMR colorectal, endometrial and ovarian cancer
MUTYH
BER of A mispaired with 8OH-dG, as well as mispairs with G, FapydG and C colon cancer

49. discovered in sporadic cancers
repeated sequences

50. DNA Repair and Clinical Syndromes: Increased
Sensitivity; Chromosomal Instability and Increased
Cancer Risk