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 costof geneticchanges

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

Outcomes of DNA repair:

Special IR Feature: Clustered Damage

Spur4 nm

2 nm

up to~ 20 bp

Repair of such a multiply damaged sitemay create DSBs

MMR Genes and CancerHereditary Non-PolyposisColon Cancer (HNPCC)• MSH2• MLH1• PMS1, PMS2

Sporadic Colon Ca• MSH2• MLH1

Sporadic Endometrial Ca• MSH2• MSH3Marti, J Cell Phys 2002

IR-induced DNA Damage is heterogeneousDamage type No./Gy/cellbase damage > 1000

single-strand 500-1000break (SSB)

double-strand ~ 40break (DSB)

sugar damage, variousDNA-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) andphosphodiesterase

DNA polymerase b adds in“C” and DNA ligase III sealsthe 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

XRCC1PARP-1 Recognition of

damage

XRCC1Ligase IIIRepair

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)

pyrimidinedimer

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 lesionssuch 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

RPAXPCXPB

XPDTFIIH

damagerecognition

helix unwinding

DNA bindingfactors

strand incisionERCC1

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 withALL overreacted to radiation therapyand was found to have a mutationin the Ligase IV gene.

Riballo, Curr Biol 1999 and JBC 2001

Measuring DNA Double-Strand Breaks1. 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/fractionAs amount of breaks > density sedimentation >

Fraction sedimentedD

NA

cont

ent

Irradiated control

Measuring DNA Double-Strand Breaks2. Neutral elution (pH = 7.4) Alkaline elution for SSB (pH = 12.2)

Fraction number

% D

NA

reta

ined 0Gy

5Gy

10Gy

20Gy

• Irradiate cells• Lyse cells on filter• Vacuum elute in neutral

pH buffer• Collect eluted buffer and

measure amount of DNAAs # of breaks > amount of DNA eluted from filter >

Measuring DNA Double-Strand Breaks3. Electrophoretic - Comet assay, pulsed field electrophoresis

Measuring defective DSBR

Pulsed-field gelelectrophoresis:

FAR = fraction ofactivity 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 end2) Re-joining of ends, but with change of sequence3) Joining of ends with other breaks/chromosomes

Cell Fate:1) Survival with genetic changes2) Apoptosis3) Mitotic death due to lethal chromosomal aberrations4) Delayed post-mitotic death or inactivation

Type of cytogenetic damage observed depends upon where in the cell cycle irradiation occursCHROMOSOME ABERRATIONS

G1 irradiationBoth sister chromatids involvedCHROMATID ABERRATIONSS or G2 irradiationUsually 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 toCarcinogenesis

Chromosomeaberrations

Small mutationsat break site

Genomic instability

Mutation of oncogenes and tumor suppressor genese.g., loss of checkpoint control, apoptotic response

Malignant cell transformation

Why are there two principal Pathwaysof DSB Repair ?

DSB Repair by Homologous orNon-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 processinghomology search Rad52

single-strand invasion

Rad51 + paralogs,Rad54, RPA, BRCA2

Uncontrolled HR may be detrimental

Up-regulated or de-regulated HRis likely an important mechanismin carcinogenesis.

Mechanisms of Loss of Heterozygosity (LOH):

gene conversion

deletion

chromosome loss

normal cell heterozygouscell

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 diversityduring V(D)J recombination

Error-prone repairof DSBs by NHR

NHR is needed for V(D)J Recombination

(6)

CE, coding endsSJ, signal joints

Grawunder & Harfst,Curr Opin Immun 2001

(1)

(2)

(3)

(4)

(4)

(5a) (5b)

Enzymology of NHR

Ku70/80

DNA-PKcsArtemis

XRCC4Ligase 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-linksDNA-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)