DNA Repair 1 :

Types of DNA damage

Radiobiology
2012

Repair of DNA damage caused by

ionizing radiation (IR) is defined by the
lesion to be repaired:
excision repair
single strand break (SSB) repair
double-strand break (DSB) repair
other repair types (ie crosslink)
DNA Damage and Chromosomal Damage

Sources of DNA damage

UV damage to skin
Replication errors generate mismatches
Spontaneous cytosine deamination
Replication fork collapse and strand breaks

Ionizations from high energy

photons/particles

Ionizing radiation
ionizes along tracks

LET is linear energy transfer

Different LET radiations have

different toxicities

LET can modify RBE

(relative biologic effect)

Maximum RBE
at 100KeV/uM

DNA damage is a complex set of lesions,

but things can be simplified:
Outcomes of DNA repair:

Accurate repair:
cell survives
without mutations

Misrepair:
cell survives
but at the cost
of genetic
changes

Inadequate repair:
cell inactivation or
death due to
mitotic death
apoptosis
permanent arrest

Special IR Feature: Clustered Damage

2 nm
up to
~ 20 bp
4 nm

Spur

Repair of such a multiply damaged site

may create DSBs

MMR Genes and Cancer

Hereditary Non-Polyposis
Colon Cancer (HNPCC)
MSH2
MLH1
PMS1, PMS2
Sporadic Colon Ca
MSH2
MLH1

Marti, J Cell Phys 2002

Sporadic Endometrial Ca
MSH2
MSH3

IR-induced DNA Damage is heterogeneous

Damage type
base damage

No./Gy/cell
> 1000

single-strand
break (SSB)

500-1000

double-strand
break (DSB)

~ 40

sugar damage,
various
DNA-DNA and DNAprotein cross links

Base Excision Repair (BER)

C-T-U-A-T
G-A-G-T-A

DNA glycosylase
AP endonuclease (APE) and
phosphodiesterase

C
G

DNA polymerase adds in

C and DNA ligase III seals
the nick

BER and Radiation Sensitivity

IR-induced base damage is efficiently repaired
Defects in BER may lead to an increased mutation rate
but usually do not result in cellular radiation sensitivity
However, one exception is mutation of the XRCC1
gene (X-Ray Cross Complementation factor 1), which
confers ~ 1.7-fold increased radiation sensitivity

Functions of XRCC1
XRCC1

Recognition of
PARP-1 damage

Repair Ligase III

of nick
XRCC1
Radiation sensitivity of XRCC1-deficient cells
may come from XRCC1s involvement in other repair
pathways, such as the repair of SSBs ....

Nucleotide Excision Repair (NER)

UV

IR
pyrimidine
dimer

Helicase
Nuclease

SSB

Polymerase
Ligase

NER and Radiation Sensitivity

IR-induced SSBs are efficiently repaired
Mutated NER genes do not cause cellular radiation
hypersensitivity
However, persistent adjacent SSBs may lead to DSBs
& thereby to cell death
Defective NER increases sensitivity to UV-induced
damage and to other lesions that affect a single strand
Germline mutations in NER genes cause
human DNA repair deficiency disorders XP CS TTD

NER: Global Genome Repair (GGR) and

Transcription-Coupled Repair (TCR)
Bulky lesions
such as UV damage

GGR
defective in Xeroderma
Pigmentosum (XP)

TCR
= repair of transcribed strand
in active genes, defective in
Cockaynes Syndrome (CS)
and in XP

Functions of XP Genes
helix unwinding
TFIIH
damage HHR23B
recognition

XPB

XPD

XPC

RPA

XPA

DNA binding
factors

XPG XPF

ERCC1
strand incision
XPC is only required for GGR - not for TCR
function of CSA and CSB is not well understood

XPD K751Q polymorphism

The Repair of DSBs

Why do we believe
that a DSB is the most important
type of DNA damage induced by IR?

DSE vs DSB

Nickloff et al Cell Res 2008

Defective DSB Repair causes cellular &

clinical Radiation Hypersensitivity
14-year old boy with
ALL overreacted
to radiation therapy
and was found
to have a mutation
in the Ligase IV gene.
LigIV+/+
LigIV-/LigIV+/-

Grawunder, Mol Cell 1998

Riballo, Curr Biol 1999 and

JBC 2001

Measuring DNA Double-Strand Breaks

1. Nucleoid sedimentation

As amount of breaks >

density sedimentation >

Irradiated control
DNA content

Irradiate cells (100Gy)

Lyse cells and layer DNA
on a sucrose gradient (5 20%)
Centrifuge at high speed
Collect fractions and
measure DNA/fraction

Fraction sedimented

 Irradiate cells
Lyse cells on filter
Vacuum elute in neutral
pH buffer
Collect eluted buffer and
measure amount of DNA
As # of breaks > amount of
DNA eluted from filter >

% DNA retained

Measuring DNA Double-Strand Breaks

2. Neutral elution (pH = 7.4)
Alkaline elution for SSB (pH = 12.2)
0Gy
5Gy

10Gy
20Gy
Fraction number

Measuring DNA Double-Strand Breaks

3. Electrophoretic - Comet assay,
pulsed field electrophoresis

Measuring defective DSBR

Pulsed-field gel
electrophoresis:
FAR = fraction of
activity released
180BR = LIG4 mutation
MRC5 = control
filled circles/squares:
transformed clones
Badie, Cancer Res 1997

DSBs can lead to Chromosome Aberrations

Immediate Outcomes:
1) No repair: loss of chromosomal end
2) Re-joining of ends, but with change of sequence
3) Joining of ends with other breaks/chromosomes
Cell Fate:
1) Survival with genetic changes
2) Apoptosis
3) Mitotic death due to lethal chromosomal aberrations
4) Delayed post-mitotic death or inactivation

Type of cytogenetic damage observed

depends upon where in the cell cycle
irradiation occurs
CHROMOSOME ABERRATIONS
G1 irradiation
Both sister chromatids involved
CHROMATID ABERRATIONS
S or G2 irradiation
Usually only 1 chromatid involved

Multiple mis-rejoining events occurring in CHO

chromosomes after G1 irradiation

tricentric

dicentrics

Chromatid deletions in CHO chromosomes

after irradiation in S or G2

Chromatid
deletion

Iso-chromatid
deletion

Combinatorial painting - limited use for rare events

Spectral karyotying (Sky)

m-FISH after irradiation

From: Dr. M. Cornforth

Inadequate DSB Repair may contribute to

Carcinogenesis
Chromosome
aberrations

Small mutations
at break site
Genomic instability

Mutation of oncogenes and tumor suppressor genes

e.g., loss of checkpoint control, apoptotic response
Malignant cell transformation

Why are there two principal Pathways

of DSB Repair ?

DSB Repair by Homologous or

Non-Homologous Recombination (HR, NHR)

HR

NHR

Gene Conversion Model of HR

"

"

HR is essential for DNA Replication

Haber, TBS 1999

The HR pathway probably has arisen to repair

- spontaneous breaks that occur during replication
- broken replication forks in order to restart replication

Execution of HR
end processing
homology search

single-strand
invasion

Rad52

Rad51 + paralogs,
Rad54, RPA, BRCA2

Uncontrolled HR may be detrimental

Mechanisms of Loss of
Heterozygosity (LOH):
gene conversion

normal cell

heterozygous
cell

Up-regulated or de-regulated HR
is likely an important mechanism
in carcinogenesis.

deletion

chromosome loss

Effects of defective HR
1. Impaired ability to repair DNA in S and G2 phase
2. Cellular hypersensitivity to IR (variable)
3. Often reduced proliferation
(because of impaired DNA replication)
4. Chromosomal instability & cancer predisposition:
- BRCA2 +/- (familial breast ca & others)
- BRCA1 +/- (familial breast ca, ovarian & others)
- BRCA1 hypermethylation (sporadic breast ca)
- mutations of Rad52, Rad54, XRCC3 and other
HR genes found in various sporadic cancers

NHR is error-prone
Intentional diversity
during V(D)J recombination

Error-prone repair
of DSBs by NHR

Mammalian genomes may tolerate

error-prone NHR, because > 90%
of the DNA sequence is non-coding.

NHR is needed for V(D)J Recombination

(1)

(4)

(2)

(5a)

(5b)

(3)
(6)
(4)

CE, coding ends

SJ, signal joints
Grawunder & Harfst,
Curr Opin Immun 2001

Enzymology of NHR
Ku70/80

DNA-PKcs
Artemis
XRCC4
Ligase IV

Effects of defective NHR

1. Impaired ability to rejoin DNA ends
2. Cellular hypersensitivity to IR
3. Impaired V(D)J recombination immune defect
For example:
SCID (severe combined immune deficiency syndrome)
4. Cancer predisposition in mice; however,
NOT (yet) linked to human cancer predisposition
(except for one leukemia pt with LIG4 mutation)
5. Developmental defects

Principal Effects of defective HR or NHR

Endpoint
Chromosomal
aberrations

HR

NHR
+

(esp. chromatid !)

Proliferative defect

Immune defect
IR sensitivity

+
-

+
++

Cellular/clinical phenotype varies with particular gene defect

Other Types of IR-induced Damage

Damage to sugar back bone
frequent IR damage
easily repaired by excisional repair mechanisms
DNA-DNA long intra-strand and inter-strand cross-links
DNA-protein cross-links
Repaired by mixture of repair mechanisms
Role for radiation sensitivity unclear
Important lesions caused by certain
chemotherapeutic drugs (cisplatinum)

Therapeutic Potential ?
Mitomycin C
Cisplatin

5-13% ICLs
5-8% ICLs

Topoisomerase I + II inhibitors
- CPT11
- Etoposide

DSBs

Combination with IR:

- additive/synergistic cell killing by increasing
DSB burden ?
- targeting tumors with certain defects in recombination ?

Summary of Key Points

IR creates a heterogeneous spectrum of DNA lesions
DSBs constitute the most dangerous type of damage
IR sensitivity correlates best with DSBs
Multiple pathways of DNA repair exist, including
BER, NER, HR, NHR, MMR
Inadequate DSB repair can either lead to
- cell death/inactivation
(due to chromosome aberrations or apoptosis), or to
- carcinogenesis (due to chromosome aberrations or
an increased rate of small mutations)