L19 - DNA damage and repair

what is the consequence of DNA damage in dividing cells?

• if DNA damage is not repaired it can lead to errors in replication or repair - leading to cell death

• if these errors are not recognised it can lead to accumulation of mutations which then can lead to cancer

what is the consequences of DNA damage in cells that are not dividing?

• blockage of transcription

• leading to reduced gene expression

• leading to functional decline of tissues and organs = ageing

what are the two types of sources of DNA damage?

1. endogenous sources

2. exogenous sources

what are endogenous sources of DNA damage?

• reactions with other molecules within the cells

• such as: hydrolysis, oxygen species, by products of metabolism

what are exogenous sources of DNA damage?

• reactions with molecules from outside of the cell (environmental)

• such as: UV, X-rays, carcinogens, chemotherapeutics

what are the two types of DNA damage?

1. endogenous DNA damage (occurring within the cell)

2. exogenous DNA damage (environment/outside the cell)

what are the types of endogenous DNA damage?

• depurination

• deamination

• methylation

• replication errors

  —> these all affect the nucleotide bases of the DNA molecule

what are the types of exogenous DNA damage?

• pyrimidine dimers + single strand breaks

  —> both affect nucleotide bases of DNA molecules

  —> single strand breaks affect 1 strand of the DNA helix

• interstrand crosslinks + double strand breaks

  —> affecting both strands of the DNA helix

what is deamination? (endogenous DNA damage)

• removal of amino group by hydrolysis - resulting in changes to the DNA bases

what is the most common deamination event?

• deamination of cytosine to uracil

• the amino group of cytosine is replaced by a carboxyl oxygen turning it into uracil

  —> chemically transforms cytosine to uracil

what happens to the strands in deamination of cytosine to uracil?

1. the deaminated cytosine has become uracil

2. the two strands get split into the old and new strands

3. the old strand has the change in the base (cytosine → uracil) and this gets reflected in the new strand

4. the new strand there’s no change in the bases

what are transition mutations?

replacement of a base with another of the same type

(purine - purine or pyrimidine - pyrimidine)

what are transversions?

replacement of a base with 1 of a different type

( purine - pyrimidine)

what are the single rings?

pyrimidines (C, U, T)

what are the double rings?

purines (A and G)

why are transition mutations more likely to occur than transversions?

because transitions are much easier and less disruptive than transversions

• because its easier to substitute a double ring (purine) for another double ring (purine) than it is to change from a single ring (pyrimidine) to a double ring (purine) structure

what is depurination (abasic site)? (endogenous DNA damage)

• when the N-glycosidic bond is cleaved where the base would be attached - this creates the abasic site

• abasic site = gap in the DNA base where the nucleotide should be

what do consequences of the abasic site and failure to undertake DNA repair result to?

• framework mutations

• on the strand where the base is missing - the depurinated base gets mutated and so the whole base pair is missing in the newly replicated DNA = very damaging to the cell

what is the reading frame?

• DNA is read in triplets (codons) during translation

• each codon codes for one amino acid

• the starting point determines the reading frame

• there are 3 reading frames on a single codon

what is a frameshift mutation caused by and what does it cause?

insertion or deletion of 1 or 2 bases

—> this shifts all the subsequent codons into a new incorrect reading frame → resulting in a whole change of proteins produced

→ disrupts normal function of proteins

what is sickle cell anemia caused by?

• single point mutation in the beta globulin gene

• this mutation in this gene causes the sickle shape of red blood cells

• caused by missense mutation which leads to missense protein

what is a missense mutation?

a single base change that causes one amino acid in a protein to be changed to a different amino acid

what does UV light cause?

• induces formation of pyrimidine dimers - causes DNA to be distorted

• can also cause interstrand DNA crosslinks and DNA protein crosslinks

what is interstrand DNA crosslinks and DNA protein crosslinks?

interstrand DNA crosslinks

→ when the base pairs crosslink between each other

DNA protein crosslinks

→ crosslink a protein to the DNA strand

both are highly toxic as they block replication + transcription

what are the two types of damage that affect the phosphate backbone of DNA structure?

• double strand breaks

• single strand breaks

what do single strand breaks affect?

• only affect 1 strand of the DNA

what are examples of things that cause single strand breaks?

1. reactive oxygen species

2. hydroxyurea + camptothecin (DNA damaging agents - chemotherapeutics)

what do double strand breaks affect?

affects both DNA strands

what are examples of things that cause double strand breaks?

1. x-rays

2. ionising radiation

3. topoisomerase II inhibitors

what are the 3 types oof DNA repair pathways?

1. Base excision repair (BER)

2. Nucleotide excision repair (NER)

3. Translesion synthesis

what does base excision repair (BER) do?

• repairs base damage (abasic sites, deamination)

• base flipping strategy to identify errors

steps of BER (base excision repair)

1. DNA glycosylase flips damaged base out and removes it creating an abasic (AP) site

2. AP endonuclease and phosphodiesterase cut the DNA backbone at the abasic site

3. DNA polymerase inserts the correct nucleotide

4. DNA ligase seals the nick

what does NER (Nucleotide excision repair) do?

• repairs damage when more than one base is involved

  (example - pyrimidine dimers caused by UV)

• excision of short patch of single stranded DNA to remove affected bases

steps of NER (nucleotide excision repair)

1. damage recognised/detected

2. excision nuclease comes along and removes a short sequence from the damaged strand

3. DNA helicase removes the strand, leaving a nucleotide gap

4. DNA polymerase fills in the gap with correct nucleotides

5. DNA ligase seals the nick to restore the strand

what does translesion synthesis do?

• translesion DNA polymerases can replicate highly damaged DNA

• these polymerases lack:

  1. poor template recognition

  2. poor substrate base selection

  3. no exonucleolytic proof reading activity

     —> they cause = most base substitutions and single nucleotide deletion mutations

what are the two mechanisms that can repair double strand breaks?

1. nonhomologous end joining

2. homologous recombination

what does non homologous end joining (NHEJ) do?

• repairs the double strand breaks by directly joining the broken DNA ends without a template

• error prone = loss of genetic info at break site

• restricted to G1 phase

how does non homologous end joining (NHEJ) work?

1. ends are resected by nuclease which allows complexes to bind to either side of the break site

2. the overhang created by the processing of the double strand break is then excised

3. the ends of the double strand breaks are ligated back together by ligase

what does homologous recombination do?

• error free

• occurs only in S phase

• uses intact sister chromatid as a template

• repairs double strand breaks using homologous sequence (sister chromatid) as a template for accurate repair

how does homologous recombination work?

1. resection of DNA ends to create single stranded DNA

2. strand invasion into the homologous template

3. DNA synthesis uses the template - the invading strand joins with the original strand

4. resolution of holiday junctions and ligation to restore the DNA

what does the cell cycle control?

DNA repair

what are the 3 stages where DNA damage is detected and acted upon to stop the cell cycle?

1. G1

2. entry into S phase

3. entry into mitosis

   to check chromosomes are lined up properly and ready to be split into the daughter cells

how is damage detected?

by upstream pathway involving two important proteins - ATM/ATR and p53

explain the steps of detecting damage (cell cycle)

1. when DNA damage occurs - ATM/ATR kinase is activated

2. this activates checkpoint 1 and checkpoint 2

3. this phosphorylates p53 - stabilises p53 and activates it and binds it to p21 gene

4. p21 renders the checkpoints of the cell cycle to make them stop the cell cycle at that specific point

5. the G1/S - CDK and S -CDK complexes inactivated

6. this prevents cell cycle progression

7. DNA repair pathways repair the damage

what happens if repair is not possible?

leads to apoptosis or diseases

what is the disease associated with NER defects?

xeroderma pigmentosum

• autosomal recessive disease

• results in a 200 fold increased risk of skin cancer

• onset of skin cancer

• lesions build up over time causing the disease

cancer and defects in double strand break repair

• majority of breast cancer is due to mutations in BRCA1/2

• BRCA1 and BRCA2 genes = essential for homologous recombination

what do BRCA2 deficient cells cause?

• increased genomic instability

• sensitivity to DNA damaging agents

• defective in homologous recombination

  → leading to predisposition to cancer

key cancer treatments

• DNA damaging agents are key to cancer treatments

• cancer cells often have faulty DNA repair

• chemo/radiation cause DNA damage

• this kills the cancer cells more than the healthy cells as the cancer cells have faulty repair

what is synthetic lethality?

• a case where the loss of function in either of the two genes alone is non-lethal but the simultaneous loss of both leads to cell death