Lecture 22 - DNA damage and repair

Question 1

Wht uis unique about DNA to other biological macromolecule ?

Answer 1

It is the only one repaired, everthing else is replaced

Question 2

What are the consequnces of DNA damage ?

Answer 2

DNA Damage -> DNA Damage to predominantly non-dividing cells -> Blocking of Transcriptionm -> reduced gene expression -> functional decline of tissues and organs = Aging

DNA Damage -> DNA Dmage to cells that proliferate -> Errors of Replication of repair -> Cell death

DNA Damage -> DNA Dmage to cells that proliferate -> Errors of Replication of repair -> mutations -> Pre-malignant field defect -> Cancer

Question 3

What are the 2 different ways cells can be under attack? Give examples

Answer 3

Endogenous - spontaeneuos in cell i.e hydrolysis, oxygen species, by-products of metabolism

Exogenous - rxns with molecules from outside the cell i.e UV, X-ray, carcinogens, chemotherapues

Question 4

What are the differences in types of DNA damage between Endogenous and Exgenous DNA?

Answer 4

Endogenous:
Depurination (Abasic sites)
Deamination
Methylation
Replication errors
(These effect ONE strand of the DNA helix)

Exogenous:
Pyrimidine dimers
Double strand breaks
Interstrand crosslinks
(Pyriminidine affects one strand the other effect two)

Question 5

What is Deamination ?

Answer 5

Removal of the amino group by hydrolysis.

Results in changes to the DNA bases

Cytosine removes NH2 molecule via hydrolysis, oxygen attaches creating Uracil

amino acid C is deanimated from C to U

This is a CG-TA point mutation

Question 6

What are the 2 different types of point mutations?

Answer 6

1. Trabnsitions - A to G or C to T. Less likely to result in aa substitutions
2. Transversions - A to C or T
   G to C or T
   C to A or G
   T to A or G

Question 7

What is depurination ?

Answer 7

When the whole base is removed.
N-glycosidic bond is a common substrate for hydrolysis left with an basic site.

Happen around 20,000 per genome a day, most common at purine bases

Question 8

What are the consequences of Depurination?

Answer 8

Frame-shift mutation
You will have one accurately syntheised strand but one has a deleted A-T nucleotide.

Frame shift mutations generate missense proteins that doesn’t function properly

e.g. sickle cell anemia single point mutation in beat haemoglobin gene causes sickle shape, cannot function

Question 9

How does UV light damage DNA?

Answer 9

DNA doesnt have flexibitly, structure is distorted.

Question 10

How does UV light cause interstrand DNA crosslinks and DNA-protein crosslinks?

Answer 10

INsterstrand crosslinks - incorrect bases pair (DNA cant unwind during replication)

DNA-protein crosslinks - form on one strand

Highly toxic

Question 11

Give examples of what causes single strand break inducers that affect phosphate in DNA

Answer 11

Reactive oxygen specices
Hydroxyurea
Camptothecin

Question 12

Give examples of what causes double strand break inducers that affect phosphate in DNA

Answer 12

X-rays
Ionising radiation
Topoisomerase II inhibitors

Question 13

What is Base excision repair ?

Answer 13

Repairs base damage using base flippping strategy to identify errors.

e.g. abasic sites and deamination

Question 13

What is Base excision repair ?

Answer 13

Repairs base damage using base flippping strategy to identify errors.

e.g. abasic sites and deamination

Question 14

What is Nucleotide excision repair?

Answer 14

Repairs damage when moprthan one base is involved
e.g. pryrimindine dimers caused by UV light

Invovles the excision of short pathces of single stranded DNA to remove the affected bases.

Question 15

What can Translesional DNA Polymerases do?

Answer 15

They can replicate highly damaged DNA

Question 16

What do Translesional DNA Polymerases lack?

Answer 16

Precision in template recognition and substrate base choice

Exonucleolytic proof-reading activity

Question 17

What do Translesional DNA Polymerases cause?

Answer 17

Causes most base substitution and single nucleotide deletion mutations. Quite error prone.

Question 18

What is double strand break repair?

Answer 18

2 mechanisms exist to repair double strand breaks

There are 2 mechanims
1. Non home ologous end joining (NHEJ)
2. Homologous Recombination

Question 19

What are the steps in Non homologous end joining?

Answer 19

Error prone
restricted to G1 phase

Usuallu results in loss of nucleoides surrounding the breaksite

Quick process

Question 20

What happens in Homologous Recombination?

Answer 20

Error-free repair
Occurs only in S-phase
Uses intact sister chromatid as a template
Double strand break is accurately repaired

Question 21

Whare are the 3 places in cell cycle where DNA damage is detected and acted upon to stop the cycle?

Answer 21

G1 checkpoint - NHEJ
Entry to S-phase
Entry into mitosis

and check for chromosome

Question 22

How is damage detected?

Answer 22

Protein kinases ATM/ATR activated and associate at the damaged site

This activates other kinases to block cell cycle. Chk1/Chk2 kinases are activated

p53 is phosphorylated and stabalised in order to bind to p21 (inhibitor of cyclin-dependent kniases)

p21 renders G1/S-CDK and S-CDK complexes INACTIVE

Prevents cell cycle progression

Question 23

What is the disease Xeroderma Pigmentosum ?

Answer 23

Predisposition disease - Xeroderma Pigmentosum

Autosomal recessive disease
High risk of skin cancer
Associated with a defect in nucleotide excision repair (UV damage)

Question 24

What are BRCA 1/2 genes?

Answer 24

Invovled in homologous recombination
80-90% of all inherited breast cancer contain BRCA 1/2

Question 25

How do BRCA2 genes cause cancer?

Answer 25

BRCA2 deficient cells cause
genomic instability
DNA damage sensitivity agents
defective in