Advanced Molecular Cell Biology - Lecture 18

describe the importance of DNA repair:

• DNA encodes the genetic instructions used in the development and functioning of all known living organisms

• The stability of DNA is therefore essential for cell survival.

• Only biological macromolecule to repair. All others are replaced

genetic stability of DNA is the most robust defence against what disease?

cancer

what are some of the ultimate consequences of DNA damage in dividing cells?

DNA damage to cells with proliferative capacity → errors in replication repair → cell death

if not cell death mutations conferring a selective advantage for clonal expansion → pre-malignant field defect → cancer

what are some of the ultimate consequences of DNA damage in dividing cells?

DNA damage to predominantly non-dividing cells → cell death

if not cell death blockage of transcription → reduced gene expression → functional decline of tissues and organs → ageing

what are endogenous sources of attack towards DNA (and proteins and lipids?

• Reactions with other molecules within the cell
• Hydrolysis, oxygen species, by-products of metabolism

what are exogenous sources of attack towards DNA (and proteins and lipids)?

• Reactions with molecules from outside the cell
• UV, X-rays, carcinogens, chemotherapeutics

what are the types of endogenous DNA damage (spontaneously occurring within the cell)?

• Depurination (Abasic sites)
• Deamination
• Methylation
• Replication errors

what are the types of exogenous DNA damage (spontaneously occurring within the cell)?

• Pyrimidine dimers
• Single strand breaks
• Double strand breaks
• Interstrand crosslinks

• Depurination (Abasic sites)
• Deamination
• Methylation
• Replication errors
• Pyrimidine dimers
• Single strand breaks
These types of DNA damage affect?

Effects the nucleotide bases of the DNA molecule and effects one strand of the DNA helix

• Double-strand breaks
• Interstrand crosslinks
These types of DNA damage affect?

effects both strands of the DNA helix

what are the DNA damage types that cause the most number of lesions per cell per day

endogenous: depurination

exogenous: peak hr sunlight

which types of DNA damage cause a much lower rate of the number of lesions per cell?

those that cause double strand breaks

define deamination:

the removal of the amino group by hydrolysis results in changes to the DNA bases

what is the most common deamination event?

deamination of cytosine to uracil

what is the result of deamination?

during DNA replication the mutated group would cause a change in the complementary base in the new strand thereby changing the sequence

Generally, transition mutations are more likely than?

transversions

why are transition (C → T and A → G) mutants more likely than transversions?

Substituting a double ring structure for another double ring structure is more likely than substituting a double ring for a single ring and vice versa

what bond is a common substrate for hydrolysis resulting in an abasic site (AP site)?

N-glycosidic bond

what is a glycosidic bond?

the bond joining bases onto the DNA backbone

how does depurination occur?

there is a lot of water around in our cells; therefore it can cleave the bases

where is depurination more frequent?

at purine bases (approx 18,000 per genome per day)

failure to undertake DNA repair results in?

mutation

what is the consequence of failure to repair depurination?

a frameshift mutation which can be potentially very damaging to the cell even more so than substitutions

how many reading frames can a protein be translated by?

3 (dependant on where the start codon is)

what type of proteins can a frameshift mutation generate?

missense

UV light induces the formation of what?

pyrimidine dimers

what do pyrimidine dimers do?

distorts DNA, joins bases together, stopping it from being flexible

UV can also cause interstrand?

DNA crosslinks and DNA-protein crosslinks

interstrand DNA crosslinks and DNA-protein crosslinks are highly toxic to the cell, why?

they block replication and transcription

give example causes of double-strand breaks inducers;

X-rays
Ionising radiation
Topoisomerase II inhibitors

give example causes of single-strand breaks inducers;

Reactive oxygen species
Hydroxyurea
Camptothecin

each type of DNA damage lesion is repaired by a specific repair pathway, name these:

• base excision repair (for deamination)

• nucleotide excision repair (NER)

• translesion synthesis

what does base excision repair do?

• repairs base damage (abasic sites, deamination)

• uses base-flipping strategy to identify errors via uracil DNA glycosylase (identifies uracil and flips it out; should only be in RNA, not DNA)

• AP endonuclease and phosphodiesterase remove the sugar-phosphate backbone where the uracil was

• now there is a single-strand gap in the DNA; DNA polymerase fills this gap with a new nucleotide

• DNA ligase comes and seals the nicks in the DNA backbone

what does NER do?

• Repairs damage when more than one base is involved

• E.g. pyrimidine dimers (caused by UV)

• Involves the excision of short patches of single- stranded DNA to remove the affected bases

how does NER work?

• Excision nuclease recognises the damage and cleaves the DNA on either side of the damage

• DNA helicase removes the damage from the backbone

• DNA polymerase fills in the gap with new nucleotides, and ligase seals the nicks in the sugar-phosphate backbone

what are the features of translesion synthesis?

• Translesional DNA polymerases can replicate highly damaged DNA

• They lack precision in template recognition and
  substrate base choice and exonucleolytic proofreading activity

• They cause: most base substitution and single-nucleotide deletion and mutations

when the sliding clamp locates an error what does it do?

Unloads the usual DNA polymerase and reloads a translesion polymerase

what are the 2 mechanisms involved in double-strand break repair?

nonhomologous end (NHEJ) joining and homologous recombination (HR)

what is the difference between NHEJ and HR?

HR uses a whole other sister chromatid to repair the break, NHEJ simply joins the strands back together

describe the characteristics of NHEJ:

• Error-prone

• Restricted to G1 phase

• Usually results in the loss of nucleotides surrounding the break site

• Important genetic information may be lost

how does NHEJ work?

• MRN creates an overhang signalling to Ku70/80 and DNA-PKcs to form a dimer across the break site, to hold the ends in place, dragging and pushing the ends together

• this forms a synaptic complex

• the 3’ overhang is chopped off by an endonuclease

• Ligase 4 seals the nick

describe the characteristics of HR:

• Error-free repair

• Occurs only in S-phase

• Uses intact sister chromatid as a template

• Double-strand break is accurately repaired

how does HR work:

• MRN processes the ends of the break, causing a 3’ overhang on either side of the break

• RPA coats the overhang, acting as a signal to subsequent proteins, BRCA1 and BRCA2, to load Rad51 onto the overhang (Rad51 nuclear filament)

• This starts strand invasion; the strand coated with Rad51 will invade the sister chromatid

• This allows the formation of a 4-way junction called the Holliday junction

• Now the sister chromatid and strand are crossed over; DNA synthesis can occur as there is now a template

• Eventually, the invading strand will rejoin its original strand, and the Holliday junction will become resolved, where the strands are separated again

• the result is accurate repair

there are 3 places where DNA damage is detected and acted upon to STOP the cell cycle, what are they?

• G1

• entry to S-phase

• entry into mitosis

activation of repair pathways is closely linked with?

levels of cyclin-dependent kinase

how is damage detected?

• ATM/ATR get activated and associate with the site of DNA damage

• This activates other kinases this activates (Chk1/Chk2 kinases) to block the cell cycle

• p53 is stabilised (via phosphorylation) and activates p21

• p21 renders the G1/S-CDK and S-CDK complexes INACTIVE.

• Thus preventing cycle progression

• DNA is then repaired

• after repair p53 is dephosphorylated and degraded by proteosomes

give an example of predisposition disease associated with defect in NER:

Xeroderma pigmentosum

what is xeroderma pigmentosum?

an autosomal recessive disease

how much is the increase in risk of skin cancer with xeroderma pigmentosum?

2000

give an example of cancer and defects in double strand break repair:

(mutations in BRCA1/2 genes causing) breast cancer

why are mutations associated with cancer?

BRCA2-deficient cells exhibit genomic instability, are sensitive to DNA-damaging agents and are defective in HR, giving a predisposition to cancer

describe the characteristics of cancer cells:

• Very fast-growing! replicate in the presence of damage

• Often have defects in repair! Unable to repair everyday DNA damage, leading to mutations

What is the Achilles heel of cancer?

DNA-damaging agents are key to cancer treatments

• a lot of cancer comes from defects in repair

• target defects in repair to improve the effectiveness of existing therapies (i.e. target HR so they become more susceptible to DNA damage)

What is the problem with targeting defects in the repair of cancer cells to improve the effectiveness of existing therapies?

relying on a fast division rate, so types of cells that replicate fast too get affected leading to many side effects

how can you improve the efficiency of existing therapies for cancer other than targeting defects in the repair of cancer cells?

Use interactions between repair pathways to increase cell death, specifically in cancer cells; this is known as exploiting synthetic lethality

describe the concept of synthetic lethality:

• You have 2 functional pathways; if either one is mutated or stops working, the cell can still function as it has another pathway to rely on

• However, if both pathways are knocked out, then the cells die

• Therefore, if you develop a drug or inhibitor of the functional pathway, there would be cell death

• This helps treat cancer cells defective in repair, i.e. BRCA1/2 and PARP inhibitors (defective in HR)

• This way there are fewer side effects, as the other cells in the body can survive