3.2 Effects of Radiation on DNA and Chromosomes Cheat Sheet (DRAFT) by Molly

Two separate long polymer chains wound around each other in the form of a double helix

The two strands are wound around each other in opposite polarity and are held together by means of hydrogen bonding which occurs between pairs of bases that are attached to the sugar units of each strand.

The two sugar- phosphate strands are wound around each other, making one full revolution every 3.4 nm (1nm = 10−9 m) in a right-­handed spiral, which is wound around a central axis, such that a major grove and a minor grove are formed.

The result is an extremely long, thin molecule which reaches up to 1 mm in length and diameter of 2 nm.

The precise sequence of bases along the DNA molecule forms the code that carries the genetic inform­ation.

Replic­ation is not continuous but occurs at a definite part or phase of the cell division cycle, the S phase.

The two new DNA molecules eventually separate to two daughter cells so that the process of replic­ation copies the genetic inform­ation in the mother cell and permits its transm­ission to two identical daughter cells.

In simplistic terms, the living cell consists of an outer cell membrane, a cytoplasm, a nuclear envelope or membrane and a nucleus. The nucleus contains the deoxyr­ibo­nucleic acid (DNA), which is the backbone of the chromo­somes and carries all the inform­ation that determines the nature of the cell and regulates its operation.

The number is linearly related to the dose over a wide range (<0.2Gy to 60,000Gy).

The number induced in oxygenated cells is three to four times that found in cells irradiated under hypoxic condit­ions.

The comple­mentary (undam­aged) single strand is used as the template for the resynt­hesis of a new length of DNA.

Non-re­paired SSBs can take part in the formation of DSBs

Double strand breaks of DNA, if not accurately repaired, can lead to cell death or the birth of an abnormal (cance­rous) cell.

They are produced by the passage of one ionising event (αD) or as a result of two indepe­ndent SSBs (βD2).

Recent measur­ements show that many can be repaired or at least rejoined.

The techniques cannot detect the presence of 'mis-r­epair' or 'error prone' repair which might be a cause of signif­icant genetic damage.

Damage to bases of DNA was first recognised in bacteria.

The number is linearly related to dose.

Thymine damage is more frequent in mammalian cells than SSBs however, there is no direct evidence showing that this forms a biolog­ically important lesion.

Normal metabolic processes:

DNA replic­ation may be associated with transc­ription errors, repaired through a number of pathways

Non-io­nising radiation, such as UV electr­oma­gnetic radiation, can cause crosslinks between adjacent bases

Heat can cause breakage of DNA molecules

DNA damage is assessed by: Comet Assay, Pulsed Field Gel Electr­oph­oresis (PFGE), or Micron­ucleus Assay.

DNA repair can also be assessed using these methods. By allowing cells to survive for some time after radiation exposure, and comparing the fragme­ntation of DNA with cells immedi­ately sacrif­iced, the amount of repair that occurs is quanti­fiable.

The slide is then immersed in an electr­oph­oresis solution and has a current applied. Undamaged DNA remains trapped in the nucleoid, whereas damaged DNA is small enough to move through the agarose gel. Once the current has been applied for a specified time, the slide is stained for DNA molecules and visualised under a specia­lised micros­cope, often with image analysis software to calculate the presence of DNA damage.

Detects differ­ences in DNA damage (and repair) at a single cell level and is commonly used for biopsy specimens from tumours.

Gel Electr­oph­oresis works because fragments of DNA have a negative charge, causing them to migrate towards the anode if a charge is run through the gel containing the DNA molecules.

Pulsed field gel electr­oph­oresis involves three pairs of electr­odes, aligned at 0°, 120°, and -120° with respect to the direction of travel. Charge is run through the sample for 10 - 60 seconds between a pair of electr­odes, with an equal time spent on each group for a net forward migration (see Figure 2.10). Larger fragments take longer to realign themselves to the changing voltage, and therefore there is increased separation of DNA fragments.

A micron­ucleus is the aberrant formation of a third, small nucleus during a mitotic division.

For this experi­ment, dividing cells are exposed to a stimuli that causes double strand breaks. Cytoki­nesis is inhibited by cytoch­ala­sin-B. The cells are then stained and examined under a micros­cope; the number of cells that contain micron­uclei are counted. About 1,000 cells need to be counted for an accurate result.

Sensing of DNA Damage: Several genes are involved in the response to ionising radiation. The actual genes involved depend on the damage inflicted as well as the stage in the cell cycle.

DSBs are difficult problems for the cell to repair. The two ends may dissoc­iate, although the histone molecules may provide some structural support. If several breaks are formed in a cell, then the cell may unite the strands incorr­ectly. The final problem is that there may not be an approp­riate template to repair the damage, partic­ularly in G1 and early S phases.

Repair by NHEJ operates throughout the cell cycle but dominates in G1/S-p­hases. The process is error prone because it does not rely on sequence homology.

Other DNA repair mechanisms such as base excision repair (BER), mismatch repair (MR) and nucleotide excision repair (NER) respond to damage such a base oxidation, alkyla­tion, and strand interc­ala­tion.

The ideal repair pathway, but it requires an undamaged copy of the DNA to function (and replace the damaged section).

The first step in HR is the detection of the DSB. This is performed by the ATM/ATR gene products, as well as the MRN complex. When activated, these proteins signal numerous other molecules (including p53), inducing a cell cycle arrest. The ends of the damaged DNA strand are processed and damaged bases are removed.

Used in all phases of the cell cycle, as it does not require a sister chromatid to function.

NHEJ also starts with recogn­ition of the strand break and signaling to the cell that damage has occurred. PRKDC plays an important role in attracting repair proteins as well as preventing the ends from dissoc­iating.

The second step involves the ligation of the two ends.

A collection of NHEJ related proteins then processes each end before ligating the ends together.

If a base is altered by any means, it causes an abnorm­ality in the shape of the DNA helix; this can be detected by glycos­ylase which removes (excises) the damaged base.

The altern­ative method of arriving at this situation is when radiation induces a SSB, which is then recognised by the PARP protein. The ends are processed (cleaned by PNK) to leave a 'clean' single strand break.

Long patching is more compli­cated, involving the removal of a section of the DNA around the single break and recons­tru­ction of the region by polymerase and ligation of the ends.

Used when a stretch of DNA has been damaged.

NER is carried out by an array of proteins. The damaged strand is detected, and incisions made up and downstream of the lesion by 5 - 10 bases. The entire section is removed and the gap in the DNA is then copied from the undamaged side of the DNA strand and ligated onto the free ends.

Occurs during DNA replic­ation, and ensures highly accurate transl­ation of DNA (important in carcin­oge­nesis).

These abnormal bases are excised with a small margin of bases on either side. The gap is then filled by DNA polymerase and the ends ligated - repair of the lesion.

Chromo­somes are thread­-like structures of DNA and protein.

A single continuous DNA molecule extends from one end of the chromosome to the other. Somewhere along the length of chromo­somes is a region that does not stain, called the centro­mere.

In cell division, the centromere divides the chromosome and each daughter cell receives one chromatid from each chromo­some. The presence of a centromere is essential, therefore, for the migration of chromo­somal pieces to the poles of the cell.

The arms of chromo­somes are subject to breakage.

Restituted chromo­somes either lose no genetic matter or so little that cells bearing them function normally enough to escape detection. Occasi­onally, however, restit­ution does not follow a break resulting in chromosome aberra­tions. Breakage of chromo­somes without restit­ution is lethal if cell division occurs after the chromosome break. Cell death, as a result of chromosome breakage, is known as 'mitotic death'. It is a principal mechanism of cell killing in radiot­herapy.

When the chromatin generates an identical strand, it replicates the break caused by the radiation. Hence, a chromosome aberration is visible at mitosis, as there are identical breaks in a pair of strands.

The radiation dose is given later in interp­hase, after DNA has been doubled, the arms are separated, and the radiation only breaks one chromatid. This leads to chromatid abbera­tions.

An example of a lethal aberration

If the 'broken ends' are within close proximity, they may rejoin to produce a dicentric chromosome with an accomp­anying fragment.

An anaphase bridge is formed when a duplicated chromosome loses both ends of a paired arm. The arms then unite, and when the cell tries to divide at mitosis it is unable to separate the fused arms.

Conven­tional Smear: the cells must be specially prepared to view the chromo­somes:

This method allows chromo­somes to be visualised under light micros­copy, where they can be counted and observed for abnorm­alities. Abnorm­alities such as transl­oca­tions are often difficult to visualise with this method, however lethal chromosome abnorm­alities are typically visible.

In-Situ Hybrid­isation: involves a a variety of techniques that have a similar process:

If required, an antibody directed against the probe is added to the cell. This antibody is capable of creating a visible effect when bound to the probe.

The antibody used in SISH causes silver atoms to collect in the region of the gene. The number of genes can then be counted using a normal light microscope (the silver appears as a dark spot).

Chromosome painting is partic­ularly useful at detecting transl­oca­tions, as the transl­ocated arm will be a different colour to the host chromo­some. FISH requires a specific microscope which can cause the molecules to fluoresce.

The detection of the presence or absence of dicentric chromo­somes in cells, partic­ularly in lympho­cytes, is a method routinely used to identify or exclude people who are suspected of being irradi­ated.

1. the exposure registered on the personal monitor, such as a thermo­lum­ine­scent dosimeter (TLD), does not appear to reflect dose received by the wearer

The dicentric chromosome is a sensitive and reliable indicator of dose in persons having recent radiation exposures because it:

2.occurs with a low background frequency;

The Lymphocyte Culture System: To calibrate dose and effect, aliquots of whole blood from normal adults are exposed to 60Co γ radiation (or x-radi­ation) to doses 0.25 to 5.0 Gy (25 to 500 rads).

The cells are then arrested (halted in the cell cycle) after division by the addition of an inhibitor and are then harvested, stained and examined under a microscope to determine the frequency of dicentric induction.

It has been found that the dose dependency for yield of dicentrics is adequately described by the linear­-qu­adratic model, Y = αD + βD2 where Y is dicentric yield (dicen­trics per cell), D is radiation dose, and α and β determine the relative importance of single and two hit events

Cells are generally regarded as having been “killed” by radiation if they have lost reprod­uctive integrity, not by whether they physically survive in the popula­tion.

Apoptosis or programmed cell death (previ­ously called interphase cell death) is a strong feature in embryo­logical develo­pment and in lymphocyte turnover.

Apoptosis occurs in particular cell types after low doses of irradi­ation e.g. lympho­cytes, serous salivary gland cells, and certain cells in the stem cell zone in testis and intestinal crypts.

This may be due to loss of genes that allow mitosis to occur or due to inability of the cell to pass on genetic material once the catast­rophe has occurred.

A rapid fall of cell numbers after irradi­ation is likely to be due to apoptosis but may also occur by mitotic death in rapidly prolif­erating popula­tions.

Necrosis is typified by cell edema, poor staining of nuclei, increase of membrane permea­bility, shut down of cell metabo­lism, and an accomp­anying inflam­matory response. Cellular necrosis generally occurs after high radiation doses.

Senescence or replic­ative senesence (RS) is a programmed cellular stress response to the accumu­lation of damage to a cell.

It silences genes necessary for the transition from G1 to S phase of the cell cycle.