clustered dna damages betsy sutherland · quantifying dna damages: pulsed field gel...

Clustered DNA Damages

Betsy Sutherland

Biology DepartmentBrookhaven National Laboratory

BMS@bnl.gov

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