DNA damage

What are the 6 causes of DNA damage?

• endogenous nucleases

• replication error

• spontaneous errors to bases

  • Deamination

  • Depurination (and depyrimidation) , and resulting ssb

• Free radical damage

  • ROS and NOS

  • Ionising radiation

• UV

• Alkylating agent

What is endonuclease?

enzymes that cleave the phosphodiester bond within a polynucleotide chain

What is exonuclease?

enzymes that cleaves the phosphodiester bond at the end of a polynucleotide chain

What is polymerase?

enzymes that add ribonucleotides to DNA strands

What is a ligase ?

fosters the joining of DNA strands together by catalysing the formation of a phosphodiester bond

Replication errors - base pairing mismatch during DNA synthsis

energy dfference between correctly paired and incorrectly paired bs is only equivalent to 1 hydrogen bond

Rare tautomeric forms of bases can stabilise incorrect bp

Error frequency of W-C base pairing alone

1-10%

Spontaneous alternation to DNA bases

• Deamination

• which one is the quickest?

The fastest is 5-methylcytosine to Thymine

Just a deamination at one site from NH2 to =O which creates thymine

Another fast one is from cytosine to uracil

Where is deamination favourable?

Deamination of cytosine is used to increase mutation rates for generation of immunoglobulin diversity

Somatic hypermutation during clonal expansion of B-lymphocytes

In lymphocytes, (activation-induced cytidine deamination) AID deaminates cystidines to yield C to U to create a U -G mismatch repair

Which two pathways induce the removal of induced c to u?

Base excision repair BER or MMR

Mutagenesis by AID in B cell - cause

If AID is targetting the immunoglobulin gene in B cell then the formation of diverse antibodies is useful

but if AID is targetting another gene, it might elicit potential tumour promoting mutations

Spontaneous deamination is not only fastest but also most…

mutagenic at 5-methylcytosine → thymine, which has 4x higher rate of deamination than cytosine.

5-Mec is an important epigenetic mark in DNA, specifically at CpG islands

methylation in CpG islands in promoters are normally hypomethylated, but their hypermethylation can silence tumor supressor gene - Rb

The deamination problem provides a reason why thymine replaced uracil in the evolution of DNA. BER recognises U in DNA as …. and replace with…

BER recognises U in DNA as a deamination product and replace with C

Spontaneous alteration to DNA bases: depurination and depyrimidination

Due to the spontaneous breakage of the … bond

Purine lost … faster than pyrimidines

Due to the spontaneous breakage of the glycosidic bond (hydrolysis), which results in an abasic site lesion

Purine lost x20 faster than pyrimidines

b-elimination can occur at abasic sites because the deoxyribose can convert to the aldehyde (open chain) form.

4. Free radicals and related ROS

The most important DNA oxidant is OH*, which is generated from the radiolysis of water, or indirectly from the superoxide (O2*-) arising from mitochondrial O2 reduction and from xenobiotic reduction

Main sites of mitochondrial superoxide generation

Complex I = NADH dehydrogenase, involves transfer of electrons from FMNH2 to Fe-S centers and to ubiquinone, which can leak electrons to O2

Complex III

Superoxide formation via redox cycling: Menadione (electrophilic) and Cytochrome P450

menadione (Vit K3) reacts with the enzyme cytochrom P450 which yields the menadione semiquinone radial, which is unstable radial

O2*-

React with itself spontaneously to form H2o2 and this reaction is greatly catalysed by SOD

Catalase convert H2020 into water

Detoxified by GSH and Peroxiredoxins

Are H2O2 and O2*- damaging to DNA, if not, how would they indirectly cause damage?

No, but they can be reduced to OH* by transition metals especially Fe2+

Fe2+ + H202 → Fe3+ + OH* + OH-

RONS play an important physological role in …

How is Hypochlorous acid (HOCl) formed in neutrophils?

defense against bacterial infection

Superoxide is generated at high concentration at respiratory burst by neutrophils

Hypochlorous acid is formd from H2o2, which is catalysed by myeloperoxidase (MPO) in activated neutrophils

Link between colon cancer in inflammatory bowel disease and HOCl

During chronic inflammation, HOCl chlorinates cytosine to form 5-chloroC, which acts 5MeC

→ cause epigenetic silencing mutation

What is peroxynitrite? how can it generate nitrogen dioxide radical and carbonate radial?

ONOO-

from O2*- + NO*

ONOO- can react with CO2 which makes NO2* (nitrogen radical) and CO3*-

Which base has the lowest reduction potential (most easily oxidised)

Guanine → 8-OxoG

• 8-OxoG does not block DNA polymerase

• Mispairs with A (from G-C to O-C) to O-A?

• End result : G to T transversion

What can be seen from the oxidative stress and liver cancer in HCV?

• 8-OxoG upregulation

• Positive correlation with hepatic iron storage

5. Most common dimer generated from UV damage?

T-T

Thymine dimer - 68%

6. Alkylating agent

What do they do to DNA bases?

Both carcinogen and anticancer

They are electrophillic compounds that add an alkyl group to nucleophilic centres (electron rich) in DNA

Can be both endogenous and exogenous

Alkylating agent: Dimethylnitrosamine

Metabolised by CYP2E1 in the liver, which turns guanine to O6-methyl G

Alkylating agent: Temozolomide

Monfunctional alkylating agent and anticancer agent

It is a prodrug that stimultanously turns into a methylating agent

Used for melanoma and glioblastoma

high oral bioavailability and good tissue distribution

MGMT cause resistance to TMZ by removing O6-MeG

Blocking BER using PARP1 inhibitor can sensitises cells to TMZ by preventing N7-MeG repair

What are some bifunctional alkylating agents and why are they useful

Cisplastin, carboplastin

Induces interstrand crosslink which are highly toxic due to interference with DNA replication

Nitrogen mustard

Bifunctional = two reactive sites on the same molecule

two alkylations on opposite strands

In a retrospective study of liver biopsies from patients infected with hepatitis C virus, levels of iron were found to correlate with the frequency of 8-oxoguanine in DNA, and with the risk of developing hepatocellular carcinoma later in life. These observations can be from

Fenton chemistry produces hydroxyl radicals that oxidise DNA bases