Lecture 11 DNA effects Flashcards
Nitrogenous bases
adenine+thymine, guanine+cytosine
DNA
Desoxyribo Nucleic Acid
What shows that the nucleus is more sensitive than cytoplasm
selective irradiation showed that the nucleus has a 100 times smaller killer dose than the cytoplasm. Radioisotopes incorporated into DNA kill cells more efficiently than in RNA or in proteins. 125I is 300 more effective in DNA than in cytoplasm or membrane.
What bases can sensitize the DNA cells
Iodine or bromine- halogenated bases and any incorporations make the DNA more susceptible to damage
3H
is useful for DNA incorporation: range 3 micrometers and E beta = 18kev.
Sensitizer
a drug that blocks DNA repair
Radiosensitive cells
that have a low number of repair enzymes
DNA width
20A or 2 nm. Double strand helix
Most common form of naturally occurring DNA damage
Apurinic/apyrimidinic sites up to 20000 per day
Each time cell divides how many DSb and SS it forms
10 and 50 000 accordingly
misrepair of DNA
non-lethal error incorporated and passed on to daughter cell (can lead to genomic instability and cancerogenesis)
DNA repair pathways
SSB: Base excision (BER) and nucleotide excision repair (NER)
BSB: non-homologous end-joining (NHEJ) and homologous recombination repair (HRR)
Crosslink repair and mismatch repair
describe base excision repair
removal and replacement of modified bases; DNA glycosylase binds DNA and removes the bases that are damaged; apurinic/apyrimidinic endonuclease breaks the backbone and polymerase fills the ga, ligase seals the break
describe nucleotide excision repair
due to UV thymine has dimer; once it is detected DN creates a bubble, enzymes cut damaged part of bubble and polymerase replaces it and ligase seals.
non-homologous end-joining (NHEJ)
just joins ends of parts separated by DSB after degradation from ends. Mostly for 1 chromatid
homologous end-joining (NHEJ)
use sister chromatid to restore and recombine the damaged and lost DNA during DSB. The damage repaired accurately using info from sister chromatid.. For 2 chromatids.
mismatch mechanism
recognitions that the pair mismatch (T+G for example), them the strand is removed and resynthesized
crosslink repair
fork-> dsb->replacement of damaged region by attaching the opposite
chromatin
beds on a string of DNA
chromosome consists of
chromatin fibers sections 1400 nm width
Complementary DNA strands are
hybridized via H-bonding and unwind under alkaline conditions.
The yield of rad damage is affected
by the macromolecular organization of DNA
Popular DNA damage assays
for both DSB and SSB: neutral and alkaline elution through filter or separation on sucrose gradients;
Comet assay: sensitive for SSB, less for DSB;
gamma 2AX focus formation at DSB.
Research DNA damage assays
DNA unwinding;
pulsed gel electrophoresis (needs high dose);
Quantification of damaged bases (for oxidative stress);
PCR
Neutral and Alkaline Elution Assays
Alkaline conditions unwind DNA to measure DSB, SSB. Neutral cond measure DSB.
pH of neutral elution
7.4
pH of alkaline elution
12.2
the amount of eluted DNA through the filter increases (in filter assay)
as # of breaks increase
Comet assay is useful because
automated, can be performed on single cell, and under neutral and alkaline condition show DSB and SSB
Pulse-Field Gel Electrophoresis (PFGE)
irradiate, isolate DNA, load in gel well; run a gel with pulses to force larger pieces of DNA into the gel (measure with fluorescence or radiolabel). As # of breaks increases the amount of DNA is gel also increases.
gamma 2AX
add phosphorus and it will glow at the DSB within a minute with a max in 10 min. Also, repair proteins are also focused there. Dephosphorization in 30 min with t1/2 = 2. Rate of repair can be assessed in 24 h
cell division stages
prophase, prometaphase, metaphase, anaphase (increased R on one side), telophase (necking), cytokinesis (divides)
Cytogenetic damage assessed
at first metaphase after irradiation
early irradiation and both sister chromatids involved
Chromosome aberration
later (or in interphase) irradiation and only 1 chromatid involved
chromatid aberration
Lesion types
deletion type and exchange type
Exchange type rearrangement
chromosome break into two chromatids: translocation - ends of chromatids connect to the different (not lethal); dicentric and fragment (lethal)- ends of two chromatids connect to each other making a ring
In chromosome and chromatid aberration inter-arm intra-change leads
to deletion and ring
In chromosome and chromatid an aberration, inter-change may lead
to dicentric and deletion
cell division
mitosis
Chromosome analyses
Conventional: staining after adding a mitosis blocking agent,
Banding: staining to produce visible bands;
FISH= fluorescence in situ hybridization: uses fluorescently tagged chromosome probes to specific chromosomes or regions of chromosomes
Micronucleus assay
small membrane-bound DNA fragments without centromere: - cultured cells with blocked metaphase nad stained for DNA
Yield of radiation-induced chromosome damage formula for one target one hit
Y= proportionality*Dose, used for deleton.
Yield of radiation-induced chromosome damage formula for multiple hit single target
Y= proportionalityDose + betaDose^2, used for lethal
Ionizing Radiation induces
base damage, SSBs, DSBs, and DNA
protein crosslinks
DNA DSB
most lethal form of ionizing radiation-induced damage, is repaired by nonhomologous recombination in the early phase of the cell cycle and homologous recombination in the late phase of the cell cycle
Defective nonhomologous recombination
leads to chromosome
aberrations and ionizing radiation sensitivity
Radiation-induced breakage and incorrect rejoining in
prereplication (early) chromosomes
may lead to chromosome aberrations
Radiation-induced breakage and incorrect rejoining in
postreplication (late) chromosomes
may lead to chromatid aberrations
0.25 Gy
The lowest single dose that can be detected readily
stable aberrations persist for many years
true
Scoring aberrations in lymphocytes from peripheral blood may be used to estimate total body doses in humans accidentally
irradiated.
true
