which dna damage is likely to be relevant in hormetic...
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Which DNA Damage Is Likely To Be Relevant In Hormetic Responses?
William A. Bernhard
Relevant In Hormetic Responses?
Dept. of Biochemistry & Biophysics, Univ. of Rochester
Shubhadeep PurkayasthaDept. of Biochem. & Biophysics, Univ. of Rochester
Jamie R. MilliganDepartment of Radiology
Current Position at NIH
Department of Radiology, University of California at San Diego
Supported by National Cancer Institute of NIH
O tliOutline1 I i i di ti t i ll l b1. Ionizing radiation triggers a cellular response by
damaging DNA
2. The high toxicity of radiation damage is directly related to formation of damage in clusters
3. Damage produced by the direct effect is comparable to that produced by the indirect effectthat produced by the indirect effect
4. Primary lesions produced by the direct effect
5. Yields of clustered damage by the direct effectg y
6. Predicted threshold for a biological response
Damage produced by ionizing radiation is different
Endogenous – 108 DNA damages per cell per year
10 mGy of radiation (~4x background) – ~2 DNA d lldamages per cell per year
Since 1000x background is lethal, there is something about radiation damage that makes it highly toxic.g g y
DNA damage is traditionally grouped into two categories
“Di ff ” d d h di• “Direct effect” damage, due to the direct ionization of DNA or from the transfer of dry electrons and holes to the DNA fromdry electrons and holes to the DNA from the hydration waters surrounding the DNA.
• “Indirect effect” damage due to the attack of bulk water radicals ( e- H. and HO. )of bulk water radicals ( e aq., H and HO ) on DNA.
DNA in aqueous solutionDNA in aqueous solution
H O + OH H+e. -
aq. OHbulk water layer
H2O●+ OH● + H+ OH●
e. -aq.
H2O.+
e.-
H2O.+
e.-
solvation shell
DNA e.-
The DNA solvation shell consists of up to ~ 20 to 22 waters/nucleotide.
What is the relativeWhat is the relative importance of the direct and indirectdirect and indirect
effects
2.6 m = length of DNA in 46 human chromosomes
200 µm = length of 46 chromosomes in metaphase
104 fold = compaction due to DNA packaging
From “Biochemistry”, Voet & Voet
Direct vs Indirect DNA DamageDirect vs. Indirect DNA Damage
30% (DNA it l ti h ll)30% (DNA + its solvation shell)
30% (Protein + its solvation shell)
40 % free water
Based on partitioning of d iti benergy deposition by
mass ratios one predicts
From “Biochemistry” , Voet & Voet
~60% direct damage~40 % indirect damage
Model – Initial Radical Ion DistributionF EPR ( l t
dRib• +
10%
From EPR (electron paramagnetic resonance) studies of solid state DNA
Base • + Pur • +
50% 40%
DNA ≡ Phos-dRib-Base
Base (mainly Gua • +)-e-
+e-
Base • - Pyr • -
(Cyt • - + Thy • - )
50% 50%
( y y )
Deoxyribose-phosphate Oxidationh i f i l t d b k f timechanism for single strand break formation
O
HO
HH
B
O
P O
OR
-O
H
.O
O
B
O
P O
OR
-O
-H+
PO
OR
O-
dRib(C1'-H).
PO
OR
O-
dRib
P O
OR
-OH
OH
P O
OR
-O
+OH-Ox
Ox-
O
HO
H
PO
OR
O-
O
H
OH
O
HH
OH
5MFur
+ Free Base
OH
dRibonoLac
PO
OR
O-
Deoxyribose-phosphate Oxidationh i f i l t d b k f timechanism for single strand break formation
O
HO
HH
B
O
P O
OR
-O
H
.O
O
B
O
P O
OR
-O
-H+
PO
OR
O-
dRib(C1'-H).
PO
OR
O-
dRib
P O
OR
-OH
OH
P O
OR
-O
+OH-Ox
Ox-
O
HO
H
PO
OR
O-
O
H
OH
O
HH
OH
5MFur
+ Free Base
OH
dRibonoLac
PO
OR
O-
Guanine Oxidationh i f 8 G f timechanism for 8oxoGua formation
Ox Ox-
HNN
O
HN
HN
O
HN
HN
O
+OH-HN
N NRH2N
. HN
N NRH2N
OH. HN
N NRH2N
O
Gua Gua(C8+OH) 8OxoGua
Guanine Oxidationh i f 8 G f timechanism for 8oxoGua formation
Ox Ox-
HNN
O
HN
HN
O
HN
HN
O
+OH-HN
N NRH2N
. HN
N NRH2N
OH. HN
N NRH2N
O
Gua Gua(C8+OH) 8OxoGua
Thymine Reductionh i f dih d th i f timechanism for dihydrothymine formation
HN
O
HN
O
. HN
O
H
Red Red+
+H+
NR
O
Thy
.
. -
NR
O HH
Thy(C6+H).NR
O HH
DHThy
H
+H+
+H
Similarly for cytosine
Cyt(C5+H)Cyt DHUraDHCyt. - .+H+
Red Red+
+H+
+H2O
NH
Similarly for cytosine
-NH3
Thymine Reductionh i f dih d th i f timechanism for dihydrothymine formation
HN
O
HN
O
. HN
O
H
Red Red+
+H+
NR
O
Thy
.
. -
NR
O HH
Thy(C6+H).NR
O HH
DHThy
H
+H+
+H
Similarly for cytosine
Cyt(C5+H)Cyt DHUraDHCyt. - .+H+
Red Red+
+H+
+H2O
NH
Similarly for cytosine
-NH3
Measurement of Clustered Damage in DNA
• Films were prepared from pUC18 plasmidshydrated to Γ = 2 5 7 5 11 5 15 and 22 5hydrated to Γ = 2.5, 7.5, 11.5, 15, and 22.5 waters/nucleotide.
• X-irradiated at 0.3-2.9 kGy/min (70 kV, tungsten target)
S d b k i ld d b• Strand break yields were measured by agarose gel electrophoresis at 4 0C.
Quantification of strand break yields in X-irradiated plasmid using agarose gel electrophoresis.plasmid using agarose gel electrophoresis.
• Form I – Supercoiled
• Form II – Open Circleindicates a single strand break (ssb).
• Form III - Linear
Dose
Form III Linearindicates a double strand break (dsb).
Open Circle
Linear
Supercoiled
Gel image of x-irradiated pUC18as a function of dose, 0-3300Gy.
Dose Response for Loss of Supercoiled and Formation of Open Circle and Linear Plasmid
0.8 SupercoiledOpen circle
p
0.6
0.8on
Open circle Linear
0.4
Conc
entra
tio
0.2
Rela
tive
C
0.0
0 1000 2000 3000
Dose (Gy)
Three types of clusters wereThree types of clusters were measured
1. Containing dRibose damage on opposing strands (within ~10 bp of each other)
2. Containing DHPyr on one strand and DHPyr or dRibose damage on the opposing strandg pp g
3. Containing 8-oxoGua on one strand and 8-oxoGua or dRibose damage on the opposing strandor dRibose damage on the opposing strand
Base damage is revealed using base excision repairBase damage is revealed using base excision repair enzymes
Type 1 ClusterType 1 Cluster
dRibose damage →ssb
X-ray
dRibose damage ssbDissolve in water
dRibose damage →ssb
DSB
DHTh DHUType 2 Cluster DHThy or DHUraType 2 ClusterX-ray
NthdRibose damage →ssb
HN
O
HHH
dRibose damage →ssb
Nth (endonuclease III):
NR
O H
enzyme that primarily recognize DHThy and DHUra
Dissolve in water
DSB
8 GType 3 Cluster 8oxoGuaType 3 ClusterX-ray
HNHN
O
OFpg
dRibose damage →ssb
Fpg (formamidopyrimidine-DNA l l )
N NRH2NdRibose damage →ssb
DNA glycosylase): enzyme that excises mainly 8-oxo-
guanine (8oxoGua)
Dissolve in water
DSB
Chemical Yields of Clusters Leading to DSB
6 G'noE
(dsb)Type 1 – dRib*:dRib*
5
noE
/J)
yp
3
4
dsb
(nm
ol/
2 G'Nth+Fpg
(dsb)
Yie
ld o
f d
Type 2 & 3 – base* :dRib* or base*
0 5 10 15 20 250
1 :dRib or base
0 5 10 15 20 25
DNA Hydration (mol water/mol nucleotide)
Yields of clustered damage in films f UC18 DNAof pUC18 DNA
Type 1: G'noEnz(dsb) 3.5 ± 0.5 nmol/J
Type 2: G'Nth(dsb) 2.3 ±0.9 nmol/JType 2: G Nth(dsb) 2.3 ±0.9 nmol/J
Type 3: G'Fpg(dsb) 3.2 ±0.9 nmol/J
Total clustered damage : direct effect only ~9 nmol/Jdirect + indirect 20 nmol/Jdirect + indirect ~20 nmol/J
S. Purkayastha, et al., Radiat. Res., in press
Predicted Threshold for a Biological Response
Human genome: 46 chromosomes = 7.8x109 bp = 5.4x1012 Da
Yield of clusters ~ 20 nmol/J
1 mGy gives ~1 cluster / 10 genomes1 mGy gives 1 cluster / 10 genomes
~1000 clusters are formed per dicentric chromosome
If damage to 1% of the cells is required for eliciting a biological response, then the threshold dose is ~ 0.1 mGy.
Isolated lesions are between 10x and 100x more prevalent than clustered lesions, but these are repaired with high fidelity.