Effects of Radiation on DNA

Irradiation of cells causes:

 cell killing

 m u tagenesis

Understanding the processes between energy deposit ion and expression of dam a ge may allow for manipul ati on of the process to e.g.,

 increase the damage t o a tu mor

 decrease the damage to norm a l tissues.

A. What is the evidence that DNA is the target?

1. Selectively irradi ate the nucleus or the cytoplasm. Results show the nucleus to be m o re sensitive than the cytoplasm.

To kill a cell the dose to the nucleus is 100 times smaller than the dose to the cytoplasm.

 Polonium needle: alpha pa rticle range ~ 4 0 µm .

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 Microbeam s capable of delivering partic les (protons or alpha particles) with µm resolution.

2. Incorporation of halogenated base analogues into DNA sensitizes cells. Iodine or brom ine

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[Tubiana, 1990]

Spatially, the halogens are sim i lar to the methyl group, thus “errors” are possi ble anad incorporation int o the DNA strand.

Incorporation makes the DNA m o re susceptible to dam a ge, including radiation damage.

3. Radioisotopes incorporated into DN A kill cells m u ch m o re efficiently than radioi sotopes in RNA or in proteins. 125 I in the DNA is 200-300 times m o re effective than 125 I in the cytoplasm or on the cell membran e .

4. Tritium is particularly useful: em its beta particles of 18 keV, range in tissue is less than 1-2 µm.

 Tritiated thym idine….labels DNA

 Tritiated uracil ….labels RNA

 Tritiated am ino acids to label proteins

 Tritiated water (uniform dist ribution) is 1000 tim es less effective than tritiated thym idine (DNA lo calization).

 [ 3 H]Thd incorporation caused ch rom o some breaks, correlating with the point of attachm e nt visualized by autoradiography.

5. Correlation between cellular DNA c ontent or chromosom e volume and radiosensit i vity, parti c ularly in sim p ler organism s such as viruses.

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[Tubiana, 1990]

6. Cells deficient in DNA repair enzymes are generally m o re radiosensiti ve. Drugs that inhibit D NA repair are sensiti zers.

B. Types of DNA Damage

Radiation can produce a v a riety of lesions in DNA

 Rupture of the strand

 Alteration to bases

 Destruction of sugars

 Crosslinks and formation of di mers

But what about the background levels of DNA damage that occur every day from “natural” sources (prim a rily oxygen)?

We have evolved sophisticated DNA damage detection and repair mechanisms to deal with this damage.

Does radiation produce unique dam a ge?

Clustered Damage

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Radiation damage to DNA

DNA Strand Break s Single strand breaks:

 Can take place at the phosphodi este r bond, or at the bond between the base and the sugar.

 A large proport i on of the singl e stra nd breaks are caused by hydroxyl radicals ( O H  ). Radical scavenging experiments have dem o nstrated this.

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[Roots, 1972]

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[Tubiana, 1990]

Double strand breaks:

 Invol ves breakage of both strands at point s less than 3 nucleotides apart (there are still questions a bout whether furthe r spacings are recognized and repaired as dsbs).

 Production by singl e particle crossi ng both strands?

 Production by two independent events?

 Can be measured by various tec hniques (e.g., sucrose gradient centrifugation)

 Double strand breaks have shown a di rect proportionality to radiation dose.

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[Prise, 1994]

** X-ray dose of ~1 Gy produc es about 1000 single strand breaks and about 50-100 double strand breaks in a typical mammali an cell.

This dose causes about 50% cell death. DSBs are not necessarily lethal.

Base changes:

Bases can be damaged or destroyed or chem ically modified by radiation. Hydroxyl radical and byprodu cts can add to bases.

Pyrim i dines (T, C) m o re sensitive than purines.

The biological signifi cance of base damag e is less th an that of strand dam a ge.

D. Techniques for measuring DNA damage

Base damage : various techniques exist for measuring the release of bases, or damaged base fragmen ts: e.g., HPLC, GC-MS, 3 H release from previously incorporat ed 3 H-thym idine, imm unological probes.

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DNA strand break s :

 Many of these techniques can me asure either ssb or dsb by manipulation of the pH.

 High pH (alkaline co nditions) will denature DNA (separate the two strands).

 Neutral conditi ons: double strands rem a in intact.

Sucrose gradient sedimentation:

 cells carefully lysed on top of sucrose gradient,

 centrifuged at high speed,

 the larger fragments will m i grat e further into the gradient

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[Tubiana, 1990]

Filter elution:

 DNA prelabeled by growing cells in radioactive DNA precursors ( 3 H or 14 C thym idine). Cells lysed gently on the filter.

 Elution through pores in a filter us ing a pum p and fraction collector.

 The am ount of DNA eluted as a func tion of tim e is proporti onal to radiation damage.

 Larger fragments elute more sl owly.

 Neutral conditi ons or alkaline condit i ons can be used.

Gel electrophoresis:

 Separation of DNA fragments according to size (and shape) in a gel of acrylam ide or agarose when exposed to an applied electric field.

 Cannot resolve very large fragments.

 A variation known as pulsed fi eld gel electrophoresis can resolve mu ch larger frag m en ts. The el ectric field is pulsed and alternated in orientation.

 Methods to quantitate the am o unt of DNA m i grating out of the well either radiolabeling or fluorescence (expressed as fraction of activity releas ed, F A R).

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Comet assay:

 Single-cell gel electrophoresis.

 Cells em b e dded in gel, lysed to re m ove proteins, then subjected to electric field.

 Smaller DNA fragments m i grate furt her making a “tail” that can be stained and viewed under a fluorescent microscope.

E. DNA rep air

Repair of dam a ge to DNA is o f central im portance to all cells, and is an ongoing process.

Existing re pair m e chanism s elim in at e m o st radiation-induced lesions.

 Restores v i ability

 But viable cells may still harb or m u tations or chromosom a l aberrations

Distingui sh repair as

 Error-free: restores DNA to its original state

 Misrepair: non-lethal errors are incorporated and passed on to daughter cells. This could lead to genom ic instability and carcinogenesis.

DNA repair is governed by a m u ltitude of genes, and executed by DNA repair enzymes.

Mutants deficient in DNA repair genes have helped elucidate t h ese co mp lex system s and their control.

Question: are there radiat ion-i nduced le sions that are unique? Lesions that the sophist icated repair system that has evolved to handle oxidative damage cannot handle?

Example of DNA repair .

Excision-repair:

 Principle mechanism of repair

 Operates on single st rand breaks, or dam a ge.

 Stepwise repair invol ves at least four di fferent repair enzym e s.

o Recognition of the presence of a lesion

o Excision of the damaged area

o Resynthesis by copying from the adjacen t strand

o Res ealing the break

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[Tubiana, 1990]

Repair of double strand breaks:

 Mechanisms not clear.

 Repair by recom b ination with t h e hom ologous st rand is possi ble.

 Large (com plex) lesions m a y not be repaired correctly

 Som e double strand breaks m a y not be repairable at all.

 Misrepair or deletions may l ead to chromo some ab errations, m u tations or carcinogenesis.

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So What Kills Cells?

Chromosome aberration s correlate wi th dose.

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[Sachs, 1993]

High LET radiation is more effect ive than low - LET for production of aberrations.

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Chromosome aberrations co rrelate wi th cell dea th.

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[Hall, 2000]

Cornfort h: observed individual cells following irradiation.

If a m i cronucleus was present in th e daughter cell, no colony was form ed.

N.B. , Ab errations presen t at mitosis, may be far removed from initial dam a ge.

Repair, cel l cycle, changes in chromatin s t ructure all can affect the results.

Chromosome aberrat i ons also show the LET dependency with a maxim u m at about 100 keV/ µ m. At high LET, the damage could be too severe for survival.

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Chromosome aberrations require a dsb.

The fact t h at the dsb do not correl ate with cell kil ling is troubling. Recent work indicates that clustered damage is involved.