37
Clustered DNA Damages Betsy Sutherland Biology Department Brookhaven National Laboratory [email protected] Biology Department
Clustered DNA Damages
Betsy Sutherland
Biology DepartmentBrookhaven National Laboratory
Biology Department
The Plan • Introduction to DNA Damage Clusters
• New dataCluster damage spectra
Endogenous clusters in smokers’ stem cells
Repair concerto
Double Strand Breaks: Critical Damages
But are they only the tip of the icebergof Critical Clustered Damages?
•Two or more lesions within 1 or 2 helical turns
•May contain– abasic sites
-oxidized bases
-strand breaks
•Hypothesized as repair-resistant, critical radiation damages(John Ward, Dudley Goodhead)
DSB
Bistranded Damage Clusters
Rick Reynolds --CPD
Does radiation really induce clusters in DNA?
??
How to measure?
Quantifying DNA Damages: Number Average Length Analysis
# damages = # molecules+T - # molecules-T
• But we really want to know damage frequency: (damages/base or /kb or /Mbp or/Gbp)
φ (damages/base) = # molecules+T _ # molecules-Tbase base
Ln = average molecular length of DNA population(bases/molecule)
φ (damages/base) = 1/Ln+T - 1/Ln-T
-T, 1 molecule +T, 3 molecules
Treatment
Measuring Double Strand BreaksDNA (cells…)No Radiation + Radiation
Isolated Lesions Clustered Damages
SSB OxyPyr DSB Clusters
Double Strand Breaks
Chen & J Sutherland, Electrophoresis, 1989B. Sutherland et al., Analytical Biochem, 1996
NeutralAgaroseGel
Measuring Clusters
-Enz + Enz - Enz
SSB OxyPyr ClustersDSB
DNA (cells…)No Radiation + Radiation
Isolated Lesions Clustered Damages
SSB OxyPyr DSB Clusters
NeutralAgaroseGel
Double Strand Breaks
Clusters
B. Sutherland et al., PNAS, 2000
Quantifying DNA Damages: Pulsed field Gel Electrophoresis,Quantitative Electronic Imaging &Number Average Length Analysis
• No specific distribution of damages required
• No radiolabelling of DNA
• Uses nanograms of DNA per measurement
• Current sensitivity = 1-2 damages/ 109 base pairs (Gbp)
JohnSutherland
John Trunk
DeniseMonteleone
DNA Damages Can Be Quantifiedin Simple & Complex Systems
Human tissue
Bacteriophage
3D human tissuemodels
Microorganisms
Human hematopoeiticstem & progenitor cells
Higher plants
Field samples
4
Now we can measure clusters IF they are present!
Does radiation induce clusters?
Experimental Design
• DNA in dilute phosphate buffer• Expose to 137Cs γ rays•Immediately transfer to enzyme buffer (HEPES)•Treat with one of the following:
-- Nth protein: mainly oxidized pyrimidines-- Fpg protein: mainly oxidized purines-- Nfo protein: abasic sites
• Agarose gel electrophoresis (native conditions)• Stain DNA with DNA-binding fluorophore• Quantitative electronic image• Number average length analysis
γ-rays Induce Clusters in DNA in SolutionS
ites/
Meg
abas
e pa
ir
OxyPyrimidine ClustersAbasic ClustersDouble Strand Breaks
0.1 1 10 100Dose (Gy)
0.1
1
10
100300
OxyPurine Clusters
0.1 1 10 100Dose (Gy)
Fpg protein
0.1 1 10 100Dose (Gy)
Endo III
0.1 1 10 100Dose (Gy)
No Enzyme Endo IV
Dose-responses on log-log plot: straight line, slopes ≅ 1:
damage cluster is formed by a single radiation hitSutherland et al., PNAS 2000
Paula Bennett
OxyPyrimidine Clusters in Human Cells
Electronic image of human DNA on electrophoretic gel
•
OxyPyrClusters
Nth:- + - +
1.6 Mbp
0.2 Mbp
DSBDSB
Unirradiated Irradiated
• Irradiate cells cold• Harvest immediately• Isolate DNA in agarose in Ar• NotI restrict• +/- Nth protein• Neutral TAFE gel• Stain with ethidium• Quantitative electronic image• Calculate number average
length• Calculate cluster frequency
Paula Bennett
Sutherland et al., PNAS 2000
X-rays Induce Clusters in Human Cells100 kVp X-rays, 28SC monocytes
0 10 20 30 40 50 60
Dose (cGy)
-10
0
10
20
30
40
50
60
Site
s/G
bp
DSB OxyPyr Clusters Abasic Clusters OxyPur Clusters
Paula Bennett
Sutherland et al., Radiation Research, 2002
0 10 20 30 40 50 60
Dose (cGy)
-10
0
10
20
30
40
50
60
Site
s/G
bp0 10 20 30 40 50 60
Dose (cGy)
-10
0
10
20
30
40
50
60
Site
s/G
bp
0 10 20 30 40 50 60
Dose (cGy)
-10
0
10
20
30
40
50
60
Site
s/G
bp
Conclusions• Radiation induces DSBs, OxyPyrimidine clusters,
OxyPurine clusters & Abasic clusters.
• Clusters are induced at very low radiation doses.
• Linear dose response for cluster induction.
• Single radiation ‘hits’ induce damage clusters.
What factors affect cluster induction?
• Radiation dose determines cluster LEVELS.
• DNA environment affects cluster SPECTRUM.(ratio of cluster types)
• Other factors?
Does Radiation Species Determine
• cluster yields?
• DNA damage spectrum?(relative levels of specific complex damages)
Where to find radiation of different LET, energy & Z?
Galactic Cosmic Rays (GCR)
high energy protonshighly charged, energetic atomic nuclei
(HZE particles)
not effectively shielded (break up into lighter, more penetrating pieces)
abundances and energies quite well known
MAIN PROBLEM:
biological effects poorly understood
but known to be most significant space radiation hazard
The Space Radiation Environment
Slide thanks to Dr. Walter Schimmerling, NASA
Galactic Cosmic Rays(GCR)
High Z“HZE”:
~85% protons~14% helium~ 1% heavier particles
0.1
1.0
10
10 2
10 3
10 4
10 5
10 6R
ELA
TIVE
AB
UN
DA
NC
E
ATOMIC NUMBER (Z)
0 5 10 15 20 25 30
C
NeMgO
FeSi
H
He
CaMn
Lighting FIRES: Full InterplanetaryRadiation Environment Simulation
NASA Irradiation FacilitiesAt
Brookhaven National Laboratory
Booster Operating Parameters:
SPECIES(Z,A)
ENERGYRANGE(MeV /
nucleon)
TYPICALDOSE
RATES(Gy/min)
H(1,1)) 100-3070 54-16Si (14,28) 90-1230 114-31Fe(26,56) 100-1100 146-43Cu(29,63) 100-1040 n.a.Au(79,97) 40-300 n.a.
TANDEMVAN DE GRAAFFS
200 ft
RELATIVISTIC HEAVY IONCOLLIDER (RHIC)
200 MeV LINAC
HEAVY ION TRANSFER
LINE
AGSRadiobiologyExperiments
AGS EXTERNAL
BEAMS NASA Space Radiation
Laboratory(NSRL)
BOOSTER
ALTERNATINGGRADIENT
SYNCHROTRON(AGS)
Slide thanks to Dr. Walter Schimmerling, NASA
Experimental Design
• DNA in dilute phosphate buffer• Expose to charged particles [H (1 GeV/n) to Fe (1 GeV/n)]
•Immediately transfer to enzyme buffer (HEPES)•Treat with one of the following:• -- Nth protein: mainly oxidized pyrimidines• -- Fpg protein: mainly oxidized purines• --Nfo protein: abasic sites• Agarose gel electrophoresis (native conditions)• Stain DNA with DNA-binding fluorophore• Quantitative electronic image• Number average length analysis
Cluster Yields Depend on the Radiation Type
Megumi Hada
Hada & Sutherland, Rad Res. 2006
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
H (1 GeV/n)
0
20
40
60
80
00
20
40
60
80
X-rays(100 kVp)
0 5 10 15 20 25 300
20
40
60
80
100
120
140
160
180
C (293 MeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
Si(586 MeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
Ti (980 MeV/n)
0 5 10 15 20 25 300
0
0
0
0
0
0
0
0
0
Fe (970 MeV/n)
0
60
120
1800
60
120
180
0 15 30 0 15 300 15 30
Dose (Gy)
Clu
ster
s/M
bpA B C
D E F0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
H (1 GeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
H (1 GeV/n)
0
20
40
60
80
00
20
40
60
80
X-rays(100 kVp)
0
20
40
60
80
00
20
40
60
80
X-rays(100 kVp)
0 5 10 15 20 25 300
20
40
60
80
100
120
140
160
180
C (293 MeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
Si(586 MeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
Si(586 MeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
Ti (980 MeV/n)
0 5 10 15 20 25 300
20
40
60
80
00
20
40
60
80
Ti (980 MeV/n)
0 5 10 15 20 25 300
0
0
0
0
0
0
0
0
0
Fe (970 MeV/n)
0 5 10 15 20 25 300
0
0
0
0
0
0
0
0
0
Fe (970 MeV/n)
0
60
120
1800
60
120
180
0 15 30 0 15 300 15 30
Dose (Gy)
Clu
ster
s/M
bpA B C
D E F
OxyPurine
OxyPurine OxyPurine
OxyPurine OxyPurineOxyPurine
Abasic Abasic
Abasic
AbasicAbasic
Abasic
DSB
DSBDSB
DSB DSB
DSB
Radiation Dose (Gy)
Radiation Type DeterminesDNA Damage Spectrum
H
C
Si Ti
Feγ
X
Particles PhotonsMegumi Hada
0.1 1 10 100 300
LET (keV/u)
0
4
8
12D
amag
e yi
eld
(Clu
ster
or D
SB
/Mbp
)
0.1 1 10 100 300
LET (keV/u)
0
4
8
LET (keV/µm)
Clu
ster
s or
DS
B/M
bp/G
y
0
4
8
12
0.1 1 10 100 0.1 1 10 1000.11.25
1.50
1.75
2.00
(Fpg
Clu
ster
s/ (N
fo C
lust
ers)
0.1 1 10 100 300
LET (keV/u)
0
4
8
12D
amag
e yi
eld
(Clu
ster
or D
SB
/Mbp
)
0.1 1
LET (keV/u)
0
4
8
0.1 1
LET (keV/u)
0
4
8
LET (keV/µm)LET (keV/µm)
Clu
ster
s or
DS
B/M
bp/G
y
0
4
8
12
0.1 1 10 100 0.1 0.1
1.50
1.75
2.00
(Fpg
Clu
ster
s/ (N
fo C
lust
ers)
Conclusions• Radiation type determines DNA
damage spectrum.
• 1 GeV/n protons produce high levels of DSBs and other clusters in DNA in solution and in cells.
Clusters in Unirradiated Cells?
Endogenous Clustered Damagesin Human Cells?
• Isolated oxidized lesions are induced in cells.• ~ 10,000 oxidized lesions induced per cell per day.• Steady state levels of ~ 2000/cell.
•Are Endogenous Clusters induced in cells?
28SC
-10
0
20
0
20
Clu
ster
s/G
bp
OxyPur OxyPyr Abasic
0
20
40
HL-60
0
20 WTK1
A MOLT-4
28SC
-10
0
20
0
20
0
20
Clu
ster
s/G
bp
OxyPur OxyPyr Abasic
0
20
40
HL-60
0
20 WTK1
A MOLT-4
Endogenous Clusters in Human Cell Lines?Most human cell lines don’t
accumulate any clusters.Two lines accumulate Oxidized Base clusters
BUT not Abasic Clusters
0.50 1.00 1.50 2.00 2.50 3.00 3.50-10
OxyPur OxyPyr Abasic0.50 1.00 1.50 2.00 2.50 3.00 3.50
-10
OxyPur OxyPyr Abasic
0
40
20
WIL2-NS0.50 1.00 1.50 2.00 2.50 3.00 3.50
-10
0
10
20
30
40
50
0
40
20
TK6
-10
10
30
50
0
40
20
WIL2-NSWIL2-NS0.50 1.00 1.50 2.00 2.50 3.00 3.50
-10
0
10
20
30
40
50
0
40
20
TK6
0.50 1.00 1.50 2.00 2.50 3.00-10
10
30
50
Clu
ster
s/G
bp
Bennett et al., FRBM, 2005
Paula Bennett
Expectation for Human Primary Cells:
• No endogenous clusters of any type
OR
• Low levels of oxidized base clusters only& No abasic clusters
Cluster Repair In Human Cells
DSB
Expectation: Attempted Cluster Repair WouldProduce DSBs
In vitro (synthetic oligonucleotides with defined cluster) : Michael Weinfeld, Susan Wallace, Peter O’NeillIn heavily irradiated cells: Susan Wallace
Reality: What do repair-proficient cells do?
Experimental Design for Repair Studies
• Irradiate cells warm.
• Post-irradiate incubation at 37oC.
• Harvest at increasing times after irradiation.
• Isolate DNA, measure DSBs & clusters as usual.
• Few if any detectable de novo early DSBs.
Georgaklias et al., NAR, 2004Bennett et al, in preparation
100
200
0.0 0.01 0.1 1 10 30
0
10 cGy
DSB
/Gbp
250
Time After Irradiation (Days)
0.001 0.01 0.1 1 10200
50
100
150
200
250
~~~ ~
~ ~
0.001 0.01 0.1 1 10200
50
100
150
200
250
0.001 0.01 0.1 1 10200
50
100
150
200
250
0.001 0.01 0.1 1 10200
50
100
150
200
250
~~~ ~
~ ~~~
~ ~~ ~
kbp
5700
4600
3500
2200
13701020785
450
22017039.922
- N P - N P - N P - N P - N P
0 d 14 d kbp
5700
4600
3500
2200
13701020785
450
22017039.922
- N P - N P - N P - N P - N P
0 d 14 d
- N P - N P - N P - N P - N P - N P - N P - N P - N P - N P
0 Gy 5 Gy Markers
0. 1 d 2 d 14 d 0 d
5 Gy
Alex G
Double Strand Break Rejoining in Human Cells
If cells don’t produce DSBs in repairing clusters, WHAT do they do?
Repair of Abasic Clusters in Human Cells
• De novo abasic clusters appear.• Presumably repair intermediates.
Time after Irradiation (D) Time after Irradiation (D)
~ PUTR clusters, 5 Gy
0 .0 1 0 .1 1 1 02 00
5 0
1 0 0
1 5 0
2 0 0
2 5 0
PUTR
-Clu
ster
s/G
bp
~ ~~ ~
~ ~
0 0 .0 1 0 .1 1 1 02 00
5 0
1 0 0
1 5 0
2 0 0
2 5 0
PUTR
-Clu
ster
s/G
bp
0 .0 1 0 .1 1 1 02 00
5 0
1 0 0
1 5 0
2 0 0
2 5 0
PUTR
-Clu
ster
s/G
bp
~ ~~ ~
~ ~
0
Nfo Clusters, 5 Gy
0.01 0.1 1 10 200
50
100
150
200
250
Nfo-
Clu
ster
s/G
bp
~~~~
~~
0 0.01 0.1 1 10 200
50
100
150
200
250
Nfo-
Clu
ster
s/G
bp
Georgakilas et al., NAR, 2004Bennett et al., in preparation
Conclusions• Cluster repair
– for repair-proficient cells, lower radiation doses:
– avoids DSB production.
– produces cluster intermediates.
The Crew• Paula Bennett• Jim Jardine• Pat Hein• Deborah Keszenman• Mamta Naidu• Brigitte Paap• Stefan Tafrov• John Trunk• Denise Monteleone
Former Lab members• Nela Cintron• Noelle Cutter• Biraj Das• Alex Georgakilis• Megumi Hada• Sunirmal Paul• Joon Song• Emily Weinert
Collaborators• John Sutherland• Jacques Laval, IGR• Murat Saparbaev, IGR• Alan Gewirtz, UPenn• Kathryn Held, MGH• Ann Kennedy, UPenn
Other Cluster Contributors
