Gene-L22-Exogneous vs Endogenous DNA agents

main endogenous and exogenous sources of DNA damage

internal:

• oxidtion

• alkylation

• hydrolysis

• deamination

• depurinationROS

exogenous

• uv radiation

• ionising radaition

• thermal disruption

• chemicals

what types of repair correspond to different types of damage?

1. replication errors→mismatch repair MMR-3’ to 5’

2. uv damage thymidine dimers→photolyase direct repair

3. crosslinkibg/carcinogenes- NER

4. alkylation→methylguanine DNA methyltransferase or BER

5. ionising radiation and replication related breaks→homologous recombination or non homologous end joining

what are the main categories of DNA repair mechanisms?

1. direct repair→ reverses damage directly by photolyase or MGMT

2. excision repair→moves damaged DNA and replaces ir-BER and NER

3. non homologous end joining→joins broken DNA ends- error protne

4. homologous recombination→uses template for accurate repair

what is direct DNA repair? how are UV induced pyramid dimers repaired?

• direct repair is the reversal of DNA damage without removing the bases: UV induced pyrimidine dimers or alkylated guanine in the O6

• photolyase-not in humans

• uses visible light energy- breaks the cyclobutane ring in thymine dimers

• restores pyrimidine bases

• in humans: NER

how does MGMT fix DNA? key features of MGMT?

• repairs akylation damage on the O6 methyguanine lesions

• MGMT- O6 methylguaneine DNA methyltransferase

• recognises and binds O6 in DNA

• transfers methyl group from the guanine to a cysteine residue in its active site

• restores normal guanine

key features:

• becomes irreversibily inactived after 1 reaction

• must be degraded and resynthesises for further repair

what is excision repair? what types exist?

• major and broad spectrum DNA repair system

1. BER- base excision repair

2. NER- nucleotide excision repair- xeroderma

3. mismatch repair MMR- lynchs syndrome- MLH1/MSH6/PMS2-3%

• Damage is recognises

• DNA is cut out

• gap is filled using complementary strand as a template

• DNA ligase seals it

how does MMR work in e.coli

• detects where there’s base mismatches and insertion/deletion loops from strand slippage. needed as DNA pol makes 1 error per 10^4 bases

e.coli

• MutS: recognises the mismatch forms a MutS2 dimer

• MutL: is a co-ordinator. binds MutS-DNA complex and links it to MutH and activates it

• mutH: nicks daughter as it is hemimethylated- recruits a helicase and endonuclease.

• MUSLH slides along DNA and liberated damaged daughter strand which is degraded

• DNA pol III- fills this gap using the template

humans:

• MutS: MSH2 and MSH6- alpha works in base substitution and small loop mm repair. beta- small loop and large loop-insertion

• mutL: MLH1 and PMS2

• MSH2/6 detects base-base mismatches and MSH2/3 detects larger loop and bind directly to the site

• this recruits MLH/PMS2- conformational change and becomes a sliding clamp. replication factor C loads DNA pol machinery and PCNA-sliding clamp . RPA stabilises ssdna

• MLH/PMS encounters RPC at 5’ and RPC loads exonuclease 1

• exo1 degrades 5’ to 3’- then inhibited by RPA as MutL no longer stimulates it

• gap is filled by DNA pol delta

how does MMR work in humans?

• MutS: MSH2 and MSH6- alpha works in base substitution and small loop mm repair. beta- small loop and large loop-insertion

• mutL: MLH1 and PMS2

• MSH2/6 detects base-base mismatches and MSH2/3 detects larger loop and bind directly to the site

• this recruits MLH/PMS2- conformational change and becomes a sliding clamp. replication factor C loads DNA pol machinery and PCNA-sliding clamp . RPA stabilises ssdna

• MLH/PMS encounters RPC at 5’ and RPC loads exonuclease 1

• exo1 degrades 5’ to 3’- then inhibited by RPA as MutL no longer stimulates it

• gap is filled by DNA pol delta

• Mismatch detection → MSH2/6 or MSH2/3

• Clamp formation → MLH/PMS sliding clamp

• Strand targeting → RFC + PCNA + RPA coordinate repair site

• EXO1 loading → RFC recruits exonuclease

• Excision → EXO1 removes DNA (5′→3′)

• Regulation → MutSα stops stimulation; MutLα inhibits EXO1

• Resynthesis → DNA Pol δ + PCNA fill gap

• Ligation → DNA ligase I seals nick

what type of DNA damage does BER fix?

• small, non bulky lesions:

• deamination

• oxidation

• alkylation

• spontaneous base loss

• single damaged bases!!!!

DOAS

what are the steps of BER? (briefly)

• DNA glycosylase recognises and removes damaged base- breaks the N glycosidic bond and makes an AP site

• AP endonuclease APE1 cuts DNA backbone 5’ to AP site: makes 5’ abased deoxyribose phosphate dRP and 3’ OH group

• dRP lyase removes remaining sugar fragment

• DNA pol inserts corect nculeotide

• DNA ligase seals the nicks in the backbone

what are the 2 pathways of BER? briefly explain both

short patch

• DNA pol beta inserts 1 nucleotide

• removes damaged deoxyribose phosphate 5’ dRP via AP lyase, DNA ligase seals the nick

long patch

• when damaged sugar is resistant to DNA pol beta lyase activity

• needs PCNA sliding clamp

• uses DNA pol epsilon or delta

• adds 2-10 nucleotides and makes a displaced DNA flap

• flap removed by FEN1

• DNA ligase seals the final nick

what does NER fix? how’s it different from BER?

• repairs BULKY helix distorting DNA damage: UV thymine dyers, large chemical adducts

• removes a short DNA segment- not a single base

main steps of prokaryotic nucleotide excision repair? 7

• UrvA2-uvrB1 scan DNA and detect distortion

• UrvB binds DNA tightly, confirmation changes and UrvA attaches

• UrvC binds and forms excinuclease complex

• UrvC cuts 5’ and 3’ and removes 12-13 nucleotides

• urvD helicase II unwinds and remove the damaged oligonucleotide

• DNA pol I fills the gap

• DNA ligase seals the nick

main steps of NER in eukaryotes? 5

• XPC-HR23 complex, XPA, RPA recognise DNA distortion and recruit machinery

• TFIIH complex with helicase XPB and XPD- create a 30bp bubblle

• XPF-ERCC1 cuts 5’ side and XPG cuts 3’ side- rmeoval of oligonucleotide

• DNA pol delta/epsilon assisted by PCNA sliding clamp- uses parent as template

• DNA ligase I fixs this

what is xeroderma pigmentosa? what pathway is damaged?

• issues with NER pathway- cannot repair DNA damage

• damage accumulates in skin cells and cause cancer

• 50% of affected children develop skin cancer by 10 without protection