BIO2089 DNA Damage & Repair

Sources of DNA damage

• Spontaneous hydrolysis

• chemical mutagens

• UV & ionising radiation

Forms of spontaneous hydrolysis

• depurination

• deamination

Depurination reaction

breaks the bonds between the base & backbone of DNA (bond between base and sugar undergoes hydrolysis)

Depurination affects

purines - loss of an A or G

Depurination results in

an abasic site (sugar and phosphate - no base, base lost by hyrolysis of bond)

Abasic site results in

base deleation in one strand and unchanged strand after replication

Deamination reaction

conversion of amine groups (NH2) to keto group (=O)

Deamination of base C results in

U

5-methylcytosine deamination results in

T

A deamination results in

hypoxanthine

G deamination results in

xanthine

Deamination results in

mutations to both strands formed after replication

Chemical mutagens

• alkylation

• oxidation

• base analogs

• intercalating agents

Alkylation

transfer of a methyl or ethyl group to bases or phosphate backbone

Alkylation of G results in

O6-methylguanine that base pairs with T

Oxidation

reaction with ROS, endogenous or exogenous

Oxidative damage to G results in

8-oxoguanine that base pairs with A

Base analogs are

analogs of natural bases incorperated during replication

Base analog 5-bromouracil replaces

T forming a tautomeric form that is stable and mispairs with G

Interclating agents are

flat molecules containg polycyclic rings that bind between bases

Intercalating agents cause

small insertions or deletions

Example of an intercalting agent

ethidium

UV radiation results in

formation of DNA photoproducts between adjacent pyrimdines

Thymine dimers caused by UV cause

helix distortions blocking replication and transcription

Ionising radiation can cause

ds breaks fragmenting the chromosome

Errors in DNA replication fixed by

strand directed mismatch repair

Errors caused by spontaneous hyrolysis reactions repaired by

• base excision repair

• direct reversal repair

Errors caused by UV and chemical abbucts repaired by

• nucleotide excision repair

• direct reversal repair

errors caused by x-rays and chemical abbducts repaired by

recombination

Direct reversal repair - damage is

directly removed

Direct reversal repair - photoreactivation

directly reverse pyrimidine dimers recognised by DNA photolyase which breaks bonds between the dimer, oginal structure reinstated

Direct reversal repair - demethylation

methyltransferase (single use protein) regonicises methyl group & breaks the bond to remove it , orgignal structure is reinstated

Base excision repair

repairs single base mispairs & damaged bases

Base excision repair mechanism

DNA glycoylase scans DNA & flips out every base, wrong base flipped out & bond hydrolysed to form an abasic site (no base) that is cleaved by AP endonuclease at 5’ & deoxyribose phophate removed by AP lyase at 3’, gap filled by DNA pol & ligase

DNA glycolases

recognise specific bases, spefific for different forms of damage

Nucleotide excision repair

repairs damage causing helix distortions (chemical & uv)

Nucleotide excision repair mechanism

repair protein complex scans DNA looking for defects , scanning complex recognises distrorion & recruit repair machinary, helicase, endonuclease

Nucleotide excision repair in bacteria

1. RNApol stalled at lesion can recruit repair proteins & disscioate

2. Scanning complex = 2 uvrA & 1 uvrB

3. damage recognised by uvrAB complex

4. uvrA dissociates, uvrB stays causing localised unwinding

5. uvrC recruited = endonuclease = cleaves upstream & downstream of damaged site

6. helicase uvrD recruited to remove ss fragment

7. DNA pol & ligase fill gap

Transcription coupled repair

actively transcribed regions of the genome are more readily repaired

Nucleotide excision repair eukaryotes

RNApol stalls at lesion, repair proteins are recruited, TFIIH acts as helicase in both transcription & NER, RNA pol restarts following repair

Strand breaks caused by

free radicles (oxidative metabolism) & ionising readiation & chemical abbucts

Homologous recombination

repairs double strand breaks

Homologous recombination requires

homologous DNA to copy from so happens in diploid or haploid after replication before mitosis, accurate repair using sister chromatid

Homologous recombination mechanism

1. exonnuclease digested ends leaving 3’ overhang

2. strand invasion of 3’ overhang into homologous duplex (D loop formation)

3. strand elongation by DNA pol

4. ends ligated to form double holiday junction

5. junction resolved

non-homologous recombination

repairs double strand breaks

non-homologous recombination is

not homology dependent, joins DNA from same or different chromosomes & error prone (deletion)

non-homologous recombination mechanism

1. KU protein (KU80, KU70 & DNA protein kinase) detectes breaks in DNA & binds to ends

2. KU:DNA complex dimerise & align duplexes drawing together 2 strands

3. DNA protein kinase is active & recruits Artemis = exonnuclease

4. Artimesis degrades ss until there is a v.small region of homology

5. polymerase & ligase join fragments

Translesion synthesis

error prone DNA synthesis across DNA lesions that stall DNA synthesis to prevent cell death (allows the DNA damage to be tolterated)

Polymerases in translestion synthesis are

base pairing independent

Translesion synthesis mechanism

1. DNA pol 3 stalled at leson sensed by SOS response detecting ssDNA

2. DNA pol IV or V activated & replace DNA pol 3

3. after synthesis across lesion DNA pol 3 resumes

4. damage left to hopefully be repaired later

Defect in strand directed mismatch repair

causes hereditary non-polypsis colorectal cancer

defects in nuclotide exsicion repair

causes xerodema pigmentosa, susceptibility to skin cancers

defects in homologous recombination

causes susceptibility to cancers - bloom’s syndrome & hereditary breast cancer