DNA Repair 1 :

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DNA Repair 1 :

Types of DNA damage

Radiobiology

2012



excision
repair


single
strand break (SSB) repair



double
-
strand break (DSB) repair



other repair
types (
ie

crosslink)



DNA Damage and Chromosomal Damage

Repair of DNA damage caused by
ionizing radiation (IR) is defined by the
lesion to be repaired:

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:

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

Outcomes of DNA repair:

Special IR Feature: Clustered Damage

Spur

4 nm

2 nm

up to

~ 20 bp

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


Sporadic Endometrial Ca



MSH2



MSH3

Marti, J Cell Phys 2002

IR
-
induced DNA Damage is heterogeneous

Damage type


No./Gy/cell


base damage


> 1000



single
-
strand


500
-
1000

break (SSB)


double
-
strand


~ 40

break (DSB)


sugar damage,


various

DNA
-
DNA and DNA
-

protein cross links

Base Excision Repair (BER)

C
-
T
-
U
-
A
-
T


G
-
A
-
G
-
T
-
A

DNA glycosylase

AP endonuclease (APE) and

phosphodiesterase

DNA polymerase
b

adds in

“C” and DNA ligase III seals

the nick

C


G

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

PARP
-
1

Recognition of

damage

XRCC1

Ligase III

Repair

of nick

Radiation sensitivity of XRCC1
-
deficient cells

may come from XRCC1’s involvement in other repair

pathways, such as the repair of SSBs ....

Nucleotide Excision Repair (NER)

pyrimidine

dimer

UV

IR

Helicase

Nuclease

Polymerase

Ligase

SSB

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

defective in
Xeroderma
Pigmentosum

(XP)

= repair of transcribed strand
in active genes, defective in
Cockayne’s Syndrome

(CS)
and in
XP

GGR

TCR

Functions of XP Genes



XPC is only required for GGR
-

not for TCR



function of CSA and CSB is not well understood

HHR23B

XPA

XPG

XPF

RPA

XPC

XPB

XPD

TFIIH

damage

recognition

helix unwinding

DNA binding

factors

strand incision

ERCC1

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

LigIV
-
/
-

LigIV+/
-

LigIV+/+

Grawunder, Mol Cell 1998

14
-
year old boy with

ALL overreacted

to radiation therapy

and was found

to have a mutation

in the Ligase IV gene.


Riballo, Curr Biol 1999 and
JBC 2001

Measuring DNA Double
-
Strand Breaks

1.
Nucleoid

sedimentation


Irradiate cells (100Gy)


Lyse cells and layer DNA
on a sucrose gradient (5
-

20%)


Centrifuge at high speed


Collect fractions and
measure DNA/fraction

As amount of breaks >
density sedimentation >

Fraction sedimented

DNA content

Irradiated

control

Measuring DNA Double
-
Strand Breaks

2.
Neutral elution (pH = 7.4)


Alkaline elution for SSB (pH = 12.2)

Fraction number

% DNA retained

0Gy

5Gy

10Gy

20Gy


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 >

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

G
1

irradiation

Both sister
chromatids

involved


CHROMATID ABERRATIONS

S or G
2

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 G
2

Chromatid
deletion

Iso
-
chromatid
deletion

Combinatorial “painting”
-

limited use for rare events

S
pectral
k
aryot
y
ing
(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

The HR pathway probably has arisen to repair

-

spontaneous breaks that occur during replication

-

broken replication forks in order to restart replication

Haber, TBS 1999

Execution of HR

end processing

homology search

Rad52

single
-
strand

invasion

Rad51 + paralogs,

Rad54, RPA, BRCA2

Uncontrolled HR may be detrimental

Up
-
regulated or de
-
regulated HR

is likely an important mechanism

in carcinogenesis.

Mechanisms of Loss of

Heterozygosity (LOH):

gene conversion

deletion

chromosome loss

normal cell

heterozygous

cell

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

Mammalian genomes may tolerate

error
-
prone NHR, because > 90%

of the DNA sequence is non
-
coding.

Intentional diversity

during V(D)J recombination

Error
-
prone repair

of DSBs by NHR

NHR is needed for V(D)J Recombination

(6)

CE, coding ends

SJ, signal joints

Grawunder & Harfst,

Curr Opin Immun 2001

(1)

(2)

(3)

(4)

(4)

(5a)

(5b)

Enzymology

of NHR

Ku70/80

DNA
-
PKcs

Artemis

XRCC4

Ligase IV

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

Effects of defective NHR

Principal Effects of defective HR or NHR

Endpoint




HR



NHR


Chromosomal





+




+

aberrations




(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





5
-
13% ICLs



Cisplatin






5
-
8% ICLs



Topoisomerase I + II inhibitors


DSBs


-

CPT11


-

Etoposide


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)