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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) - PowerPoint PPT Presentation
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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 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)
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