Ch. 2 DNA strand breaks

Radiation effects on DNA

-Base Damage -Single strand break -double strand breaks -crosslinks consisting of intrastrand and interstrand links -stickiness or clumping of chromosomes (due to protein alteration)

Telophase

-Chromatids arrive at poles and begin to uncoil -Nuclear membranes reform and nucleoli -chromosome coils unwind until the nuclear regains the appearance of interphase -cell divides through cytokinesis

Metaphase

-Chromosomes move to center of cell -spindle forms -centromeres divide and dissolve

Ring formation

-breaks in each arm of centromere of the same chromatid -sticky ends join to one another to form a ring and fragment -upon replication forms two overlapping rings and 2 acentric fragments

The interaction of 2 double strand breaks may result in

-cell killing -carcinogenesis -mutation

Prophase

-chromatin thickens -coiled DNA condenses and wraps around the histones and condenses into light coils -centromere appears -nuclear membrane disappears

Anaphase Bridge

-damage occurs to sister chromatids which then join -during anaphase the centromere of each is pulled to either pole of the cell with the joined portion creating the bridge -produces a dicentric chromatid and 1 acentric fragment

Single Cell Electrophoresis advantageous for:

-detecting differences in DNA damage and repair at the single cell level. -biopsy specimens of tumors

Chromatid aberrations

-occurs during interphase to a single chromosome arm. -anaphase bridge

Chromosome structure

-paired chromatid -coiled chromatin -uncoiled 'beads on a string' -double helix

The yield of Double stand breaks in irradiated cells is approximately

0.04 times that of Single strand breaks

D0 dose

A dose of radiation that induces an average of one lethal event per cell leaves 37% still viable. Usually between 1-2 Gy for mammalian cells.

Attached to the backbone of each strand are four bases:

Adenine, guanine, thymine, and cytosine.

Anaphase

Chromatids move along spindles to the cell poles to get copies of each chromatids on either side of the cell

Both of the assays are cell based where

DNA in cells is much more resistant to damage by radiation than would be expected from studies on free DNA. This is due to: -the presence in cells of low MW scavengers that mop up some of the free radicals produced by ionizing radiation -the physical protection afforded the DNA by packaging with proteins such as histones

D0 Dose

Dose needed to provide enough DNA damage to cause an average of 1 lethal event/cell. There may be cells that do not undergo a lethal event and others that undergo multiple

If the breaks are opposite from one another or separated by only a few base pairs, it may lead to a

Double strand break

If the alkaline buffer is changed to a neutral pH this method of single cell electrophoresis can be used to measure

Double strand break repair

G2 phase

Has DNA replicated, is the cell big enough, and the environmental suitable

Cell Cycle (Mitosis)

Interphase Prophase Metaphase Anaphase Telophase

Double strand breaks of D0 dose

40 (results in lethal events)

base damage of D0 dose

>1000 (easy to repair)

Pulsed Filed Gell electrophoresis (PFGE)

Most widely used method to detect the induction and repair of DNA double strand breaks.

Direct Action

Occurs when damage to the DNA occurs by direct interaction of radiation with the DNA

Dicentric formation

Occurs when there is exposure in early interphase. Fusion between breaks on 2 chromosomes Sticky ends at each break combine After replication products are a dicentric chromosome and 2 adjacent fragments

95% of the energy deposition of x and gamma rays appear as

SPURs

If a modes dose is used only

Single strand breaks occur

When a dicentric cell moves into the S phase

The cell will duplicate whatever DNA happens to be there no matter what it is attached to. As this duplication occurs, there will be two centromeres as well as two DNA fragments. This is lethal

The radiation induced break is in a single strand of chromatin, during the DNA synthetic phase that follows, this strand lays down an identical strand with the break next to itself and replicated the break that has been produced by radiation. This leads to a

chromosome aberration visible at the next mitosis.

Double strand breaks are the most important lesions produced in

chromosomes by radiation

The damage of double stand breaks is more dire in its opportunity to repair because

complementary bases on either side of the molecule were removed. May or may not be a big deal. There will be a mutation that may or may not be expressed; depends on the gene damaged.

SPUR

contains up to 100eV of energy and involves on average 3 ion pairs.

Chromosomal and Chromatid aberrations result from

damage during early interphase

Single strand break

delete base and DNA strand

The mechanisms used to repair ionizing radiation induced base damage are

different from the mechanisms used to repair DNA DSBs.

Depending on the stage of cell cycle..

different repair pathways are used to repair DNA damage.

D0 dose can be

distinct or relative

DNA structure

double helix of alternating sugar and phosphate groups (backbone) having 1 of 4 bases attached to the deoxyribose sugar

Free radicals and direct ionizations may form

double strand breaks

Chromosomal aberrations occur

early in interphase before replication of DNA before synthesis cycle has occurred.

The chromosomes appear to be pulled toward the poles of the cell by

fibers attached to the centromeres

The spindles are composed of

fibers that cross the cell, linking the poles

The backbone of DNA is made up of molecules of sugar and phosphates, which serve as a

framework to hold the bases that carry the genetic code

PFGE is based on the electrophoretic elution of DNA

from agarose plugs within irradiated cells have been embedded and lysed. The fraction of dan released from the agarose is directly proportional to dose.

Actively translating genes (Susceptible regions)

genes within the nucleus that are used frequently to produce products that the cell needs to survive. If damage occurs, it can be bad. May cause cell death, or mitotic death.

This assay of single cell electrophoresis has

high sensitivity for single strand breaks

The DNA is wrapped around

histones

DNA consists of two stands held together by

hydrogen bonds between the bases

The largest part of the life of any somatic cell is spent in what phase?

interphase

G1 phase

is the big enough and the environment suitable

Telophase is the

last phase of mitosis

The quantity of DNA in the nucleus doubles as each chromosome

lays down an exact replica of itself next to itself.

Base Damage

leads to a change or loss of a base. May or may not be a big deal. Depends on how rapidly it is repaired and what the particular situation is. A damaged base constantly occurs. Our cells have advanced repair mechanisms

BLOBS

less frequent for x and gamma rays; contain an average of 7 ion pairs with an energy range of 100-500eV

Ring formation is

lethal

The incidence of most radiation induced aberrations is a

linear quadratic function of dose.

To assess DNA single strand breaks and alkaline sensitive sites..

lysis is performed with an alkaline buffer.

Certain regions of DNA, particularly actively transcribing genes, appear to be

more sensitive to radiation

If the repair of a single strand break is incorrect it may result in a

mutation

If both strands of DNA are broken, but the breaks are well separated repair will

occur again readily.

Single strand breaks are repaired using the

opposite strand as a template.

Successive divisions lead to

progressive shortening and after 40-60 divisions, the telomeres in human cells are shortened dramatically. Vital DNA sequences began to be lost.

Indirect action

radiolysis products (free radicals, ions) have an opportunity to react with the DNA molecule

The quantities of strand breaks can be measured in

relevance to the amount of radiation applied.

When going into S phase, the DNA is

replicated in the formation of two rings that are attached to a single centromere.

G0 period

resting phase; something in DNA tells the cell to go into this phase. Stays until it is told to turn on.

PFGE allows separation of DNA fragments according to

size with the assumption that DNA double strand breaks are induced randomly.

Damage to dna is caused by

some deposition of energy into the cells or DNA itself. (if it gets hit, it gets hurt)

Mammalian cells have developed

specialized pathways to sense, respond to, and repair base damage, SSBs, DSBs, sugar damage, and DNA-DNA crosslinks.

S phase

start duplicating chromosomes

DSB broken ends appear to be

sticky

Cancer cells and stem cells have the enzyme

telomerase which prevents this from occurring

Each time a normal somatic cell divides,

telomeric DNA is lost from the lagging strand because DNA polymerase cannot synthesis new DNA in the absence of an RNA primer.

IN Single cell electrophoresis, if the cells are undamaged

the DNA remains compact and does not migrate

In single cell electrophoresis, if the cell has endured double strand breaks..

the amount of damage is directly proportional to the migration of DNA in the agarose.

A lethal event may cause

the death of the cell but it is in reference to mitotic death. (cell will not be able to divide)

The amount of dose can be determined based on

the presence of these formations.

the potential for the formation of dicentrics or rings is based on

the quantities of dose received and how the cell attempts to repair themselves.

Once the chromosomes are stabilized at the equator of the cell...

their centromeres divide and metaphase is complete.

If a dose of radiation is given later in interphase after the DNA material has doubled and the chromosomes consists of 2 stands of chromatin,

then the aberrations produced are called chromatid aberrations.

When the chromosome becomes visible at mitosis,

they are present in the duplicate

pyrimidines

thymine and cytosine

Dicentric means

two centromeres

Dicentric formation occurs between

two sister chromatids on the same chromosome. There are two fragments, one with centromere still attached to it.

The energy from ionizing radiation is not deposited

uniformly in the absorbing medium but is located along the tracks of the charged particles set in motion

Chromosomal and Chromatid abberations differ by

what time the cell is aberrated

Specific sequences (Susceptible regions)

areas of regulatory genes (instructional genes). May cause unregulated division (cancer)

A break that occurs in a single chromatin arm after chromosome replication and leaves the opposite arm of the same chromosome undamaged leads to

chromatid aberrations

DSBs can lead to

chromosomal aberration that present problems at cell division

Mammalian cells D0 dose =

1-2 Gy

Interphase contains the following intermediates

1. G1 2. S phase 3. G2 phase 4. G0 period (resting)

The broken fragments of DSBs behave in different ways

1. The breaks rejoin and normal mitosis results 2. The breaks fail to rejoin and five rise to an aberration, which is scored by deletion at next mitosis 3. the broken ends rejoin to give rise to chromosomes that appear distorted in the next mitosis.

Single strand breaks of D0 dose

1000 (easy to repair)

The number of DNA lesions per cell detected immediately after a dose is approximately:

Base damage > 1000 SSBs 1000 DSBs 40

Spurs have twice the diameter of a

DNA double helix.

Mitosis Checkpoint

are the chromosome's aligned

Chromosomal aberrations result if

a cell is irradiated early in interphase before the chromosome materials have duplicated.

Chromosomal/Chromatid aberrations

a chromatin replication completed that includes the break

Purines

adenine and guanine

The backbone of each DNA strand consists of

alternating sugar and phosphate groups.

D0 dose can be applied to

any cell type, not just mammalian.

Attached to each sugar molecule is a

base

Uncoiled DNA wrapped around histones

can be positioned as coiled chromatin

Telomeres

cap and protect the terminal ends of chromosomes. Specific base sequences that acts to prime the replication of DNA during each cell division. There is a limited amount of telomeres.

Double strand breaks

cause multiple breaks and possible deletions.

senescence

cell cannot divide further

DNA is the principle target for the biological effects of radios including:

cell killing, carcinogenesis, and mutation

Ring fragments have no

centromeres and probably will be lost at mitosis because they will not be able to be pulled to either poll of the cell.

Breaks in DNA strands can be caused by

charged particle tracks and by the chemical species (such as radicals) produced.