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

Non-homogeneous energy d i i d TRACKdeposition produces a TRACK

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

Research Objective

DAMAGEDAMAGE

Type

Yield

Spatial distributionSpatial distribution

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)

Formation of linearized pUC18 DNA

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.