ES2 DNA REPAIR AND GENE EDITING

Turcot syndrome type 1

Caused by mutations in DNA mismatch repair genes → impair the cell’s ability to correct replication errors (base mismatches and insertion/deletion loops) → DNA errors accumulate during cell division

Leads to microsatellite instability (MSI) and a high mutation rate → Colorectal tumours, brain tumours (glioblastomas)

DNA damage

Chemical modification of bases (Tobacco smoke)

UV exposure

ROS

Cosmic radiation

Errors in DNA replication (~70 - 150 base changes per generation)

Tobacco smoke

Benzylpyrene (Chemical carcinogen) and major component

Intercalates DNA and distorts double helix by adding to Guanine

UV exposure

Dimerisation of adjacent thymine bases

Thymine dimer causes bulge in double helix

Synonymous/Silent mutation

Doesn’t change aa

Missense mutation

Changes aa

Nonsense mutation

changes aa to STOP

Frameshift (in/del) mutation

changes all aa after that point

Deletion mutation

Skips a stretch of aa

Splice site change

changes how mRNA is spliced

Spontaneous deamination of cytosine

Amine replaced by water (C → U)

If U in DNA → signal to correct

Methylation for transcriptional regulation (C → T)

Mutation remains after replication

DNA polymerase

Proof reading

5’ - 3’ polymerase activity

3’ - 5’ exonuclease activity (Distorted DNA strand moves backwards into exo site for correction)

Base excision repair/BER

Repairs deaminated Cytosines and oxidation products

DNA glycosylase - Specifically recognises Thymine bases where a Cytosine should be leaving an abasic site

APEI endonuclease - Cleaves abasic site

DNA polymerase ß & DNA ligase - Removes backbone, replaces nt, and seals

Mismatch repair/MMR

Repairs small mismatches and slippage of repeated DNA on newly synthesised DNA

MutS - MSH enzymes recognise mismatch through bulge in DNA structure and nicks

MutL & DNA exonuclease - MLH endonuclease splits damaged strand and segment removed containing mismatch

DNA Polymerase 𝜹 & DNA ligase - Repairs gap and seals

Nucleotide excision repair/NER

Repairs larger DNA errors (larger region than MMR)

Done through global genomic repair NER or transition coupled NER

TFIIH (helicase) - Opens strand

RPA ssDNA - Binding protein to potect bubble

XP endonucleases - Cut damaged strand (23 - 32 bp)

DNA polymerase & DNA ligase - Replaces DNA and seals

Global genomic repair NER

Corrects broad range of lesions

XPC + Rad23B enzymes recognise helix distortion

Transition coupled NER

Stalled transcription by RNA polymerase acts as signal

Double stranded DNA breaks/DSBs

Replication fork collapse during replication (5 - 10% of cells in culture have it)

Ionising radiation and chemical damage

Repaired by non-homologous end joining or homologous recombination repair

Non-homologous end joining

Occurs throughout cell cycle but error prone

DNA-PK + KU protein complex - binds DSB ends

Artemis (5’ - 3’ exonuclease) - trims sticky ends

DNA ligase - joins ends together

Homologous recombination repair

Uses other chromosomes as accurate genetic reference to accurate repair break

Only occurs during S and G2 phase (Chromosomes are adjacent to each other)

EXO1/DNA2 exonucleases - trims DSBs to create sticky ends

Rad51 - helps overhanging strand to invade complementary strand

DNA polymerase - extends invaded strand and displaces complementary strand which does the same to the other break, and extends both strands

DNA ligase & Resolvases - seals and cuts Holliday junctions (crossover structures) and joins ends

CRISPR-Cas9

Clustered regularly interspaced short palindromic repeats

Originated as bacterial defence against viral pathogens

Bacterial Cas9-tracrRNA-crRNA complex finds and cuts viral DNA (must include PAM (protospacer adjacent motif) sequence)

CRISPR-Cas9 as a tool

Uses guideRNA (crRNA and tracrRNA linked via linker loop) with a sequence of target DNA to create a DSB

Homology-directed repairs DSB using DNA template with desired sequence

Gene editing for HIV prevention

European descent carry CCR5 mutation (1% homozygous for immunity)

First genetically engineered human babies (one heterozygous, one homozygous)

Gene editing for cholesterol

VERVE101 - mRNA in a lipid nanoparticles via intravenous infusion

Edits A-T to G-C within splice donor site to silence PCSK9 (regulates LDL receptors) in hepatocytes