Lecture 3 - DNA Damage and it's Repair Part I

ONCOL 335 - Radiobiology. University of Alberta

How do we know that DNA is the critical target of radiation

• theory

• experimentation

what experiment was done to prove DNA is the critical target

selective irradiation of the nucleus or the cytoplasm shows that the nucleus is more sensitive than the cytoplasm

• to kill a cell, the required dose to the nucleus is 100 times less than dose to cytoplasm

what experiment was done to prove that DNA is the critical target

Polonium rods (high LET) were put into both the cytoplasm and nucleus

• 250 Gy to cytoplasm had no effect on cell proliferation

• 10 Gzy to nucleus resulted in cell death

Radioisotopes incorporated into DNA kill cells …

more efficiently than radioisotopes in RNA or proteins

cells deficient in DNA repair enzymes are more …

radiosensitive

drugs that inhibit DNA repair are …

sensitizers

the bigger the genome, …

the greater the probability the radiation hits the nucleus

• easier to shoot an elephant than a pigeon

what is more radioresistant, the human genome or yeast genome

the yeast genome

• it is much smaller than the human genome, making it harder to hit the nucleus

human cells have roughly ___ base pairs, ___ protein coded genes, and ___ non-coding genes

6 billion, 20 000, 26 000

Structure of DNA

• phosphate group

• sugar (deoxyribose)

• nitrogenous base

how is the bond between C-G different than between A-T

C-G bond is made up of 3 hydrogen bonds, A-T is made up of 2 hydrogen bonds

What is the difference between a ribose sugar and a deoxyribose sugar?

deoxyribose in DNA has a -H bonded to the 2’ carbon, while ribose in RNA has an -OH to the 2’ carbon

what is the structural difference between thymine and uracil

thymine has an extra methyl group

what are larger, pyrimidines or purines?

Purines (A and G)

DNA polymerase adds bases ____

5’ to 3’

How can DNA polymerase check if it added the correct base pair

it can check how many hydrogen bonds are on the base pair

External Sources of DNA damage

UV light (non-ionizing), chemicals, radiation

internal sources of DNA damage

ROS and DNA replication errors

most common DNA replication error

DNA polymerase puts on a ribonucleotide instead of a deoxyribonucleotide

5 types of DNA damage

1. SSB

2. DSB

3. Base Modifications (addition of DNA adduct)

4. Interstrand crosslinks

5. Base mismatches, indels

What DNA repair pathway fixes SSBs

BER

What DNA repair pathways fixes DSBs

HR and NHEJ

What DNA repair pathways fixes base modifications

NER or direct reversal

What DNA repair pathway fixes inter-strand crosslinks

FANC

What DNA repair pathway fixes base mismatches and indels

MMR

What type of DNA damage can result of ionizing radiation

all 5 types: SSBs, DSBs, Base modifications, inter-strand crosslinks, base indels/mismatches

What is the DNA damage response?

DNA repair + all other things cell does when DNA damage occurs

• increases energy, shut town transcription, stop cell cycle, etc

1 GY of x-ray ionizing radiaiton causes over ___ damaged bases, ___ SSBs, and ___ DSBs

1000, 1000, 30-40

What type of DNA damage is the least common but the biggest problem for the cell

double stranded breaks

Lethal damage - definition

irreverisble, irrepairible, damage leads to cell death

Sublethal damage - definition

repaired in hours, if a second dose is given, can interact with more damage to create lethal damage

potentially lethal damage - definition

can be modified by the post-irradiation environment to become lethal

what is the consequence of unrepaired DNA damage?

Chromosome damage

What are the three ways we can test for chromosome damage after irradiation?

1. Karyotyping

2. FISH

3. Micronuclei test

Karyotyping

look at chromosomes after staining them

• under a microscope we may be able to see chromosome break or unrepaired damage

FISH

Fluorescent in-site hybridization

• painting chromosomes to see if DNA chromosome fusions occur

micronuclei test

a third nucleus is formed during anaphase of mitosis after a chunk of the chromosome falls off after irradiation

• chromosome fragments develop nuclear membranes and form a third nucleus

radiation ___ the amount of miconuclei

increases

how are micronuclei treated?

cytochalasin B: allows nuclear division, but stops cell division

Aberrant Chromosomes

broken ends of chromosomes rejoin with other broken ends to generate rings, dicentrics, and translocations

Dicentric Chromosome Aberrations

the joining of 2 broken chromatids in different chromosomes

• can be used as marker for radiation exposure

when can dicentric chromosome aberrations be detected?

after replication

examples of lethal aberrant chromosome damage

ring chromosomes, dicentric chromosomes, anaphase bridge

• rings and anaphase bridge cannot be pulled apart in mitosis

examples of non-lethal aberrations

small deletions

symmetric translocations

most chromosome aberrations lead to

cell death

Translocation

reciprocal exchange of chromosmal fragments between two or more chromosomes

symmetric translocation

may be compatible with a normal phenotype

• may have reproductive failures

imbalanced translocation

offspring may receive only one of the rearranged chromosomes, becoming genomically incomplete

• lead to miscarriage or impacted physical/mental development

What are the single stranded ends of DNA called?

telomeres

how does the cell know that a telomere is normal and not a DSB?

The cell's telomeres has a long set of repeats, and if they are flipped over on themselves, they can complement the other strand

• So technically the DNA ends can pair with it's own strand, so it doesn't look like a DSB

with high LET, do we get direct or indirect ionization actions?

direct action; electron damages the DNA strand directly if it lies in the direction of the electron track

with low LET, do we get direct or indirect ionization actions?

indirect ionization

• electron hits radical species first, then species damages DNA

In low LET indirect ionization, electrons hit secondary molecules, leading to

clouds or pockets of ionizations on DNA molecules

two types of ionization clouds

Blobs and spurs

• blobs larger than spurs

What damage products on DNA are a result of UV damage

Thymine Dimers (T-T) or photoproducts

is UV damage easy for the cell to recognize?

yes, because the T-T dimers will kink the strand

What are the four general steps of DNA repair

1. recognize DNA damage

2. remove damaged nucleotides

3. fill the gap with DNA synthesis

   • polymerase needed

4. Seal the gap

   • ligase needed

What DNA repair mechanism is used to repair UV radiation damage

NER

NER - Step 1

DNA damage is recognized by XPC

NER - Step 2

excision of damaged DNA base pairs by XPF and ERCC1

NER - Step 3

New DNA is synthesized and filled into gap by polymerase beta

NER - Step 4

DNA gap is selaed with Ligase I

Mutations in the XP genes cause?

xeroderma pigmentosum and Cockayne syndrome

why is BER DNA damage more difficult to recognize?

it is harder to see a single nucleotide that was damage

BER - Step 1

DNA damaged recognized by DNA glycosylase

BER - Step 2

Damaged DNA is removed by APE1

BER - Step 3

DNA gap is filled with DNA polymerase beta

BER - Step 4

Gap is sealed with Ligase III and XRCC1