Week 10: DNA Damage, Repair + Genome Editing

DNA damage

chemical changes to nitrogen rich bases

• N-rich bases not inert → reactive

  • can lead to changes in BP potential → mutations

  • can lead to double-strand breaks → genome instability

  • can be repaired or cells undergo cell death (apoptosis)

• caused by

  • oxidative damage

  • spontaneous deamination

  • loss of base

  • replication errors

  • UV exposure = photo-cross-linking

  • chemical exposure = alkylation/methylation damage

spontaneous deamination

cytosine loses exocyclic amino group → turns into uracil

• uracil chemically similar to thymidine = can base pair w/ adenine

• leads to AT mutation if unrepaired

• recognized as foreign in DNA + removed

  • why DNA has thymine rather than uracil

• other bases can also undergo reaction

cytosine deamination

turns into uracil

adenine deamination

turns into hypoxanthine

• hypoxanthine = non-coding

  • recognized by cell as damage → removed

5-methylcytosine deamination

turns into thymine

• _______ = modified base in DNA w/ high frequency in patches

  • enhances/represses transcription = controller

• cell can’t tell that base is incorrect → won’t remove

guanine deamination

turns into xanthine

• xanthine

  • recognized by cell as damage → removed

depurination

hydrolysis of N-B-glyosyl bond b/w base + pentose

• H2O comes in + hydrolyzes bond = base floats away

• phosphodiester bond still stuck in DNA

  • creates AP (apurinic/apyrimidic) site = abasic

• info void region generated

• more common w/ purine

  • double-rings = good-leaving group

    • why AMP is used as activator

oxidative damage

reactive oxygen species damage DNA

• mitochondria = oxidative compartment → generates lots of energy via oxidative phosphorylation

• hydroxyl (OH) radicals responsible for most damage

• cells have defense system to destroy reactive oxygen species

• eg. guanine → 8-oxo-guanine

  • anti-conformation usually favoured

  • new steric clash b/w carbonyl + ribose oxygen = syn-conformation favoured

    • ⬆ Hoogsteen base-pairing b/w G + A

  • after replication → A-T pair

endogenous damage

spontaneous

• deamination

  • changes H-bonds → drives mutations after replication

• loss of base

  • generates abasic sites → loss of info

• oxidative damage

  • changes from anti → syn-conformation

  • changes base pairing

exogenous damage

DNA damage caused by external factors such as chemicals, radiation, or infectious agents

• photo-cross linking from UV radiation

  • covalent bonds form b/w bases on same strand instead of H-bonds w/ base across → lesion

• alkylation from chemical exposure

  • addition of alkyl (eg. methyl) groups to base → affects base pairing

photo-cross-linking

caused by UV damage or radiation

• UV

  • cyclobutane pyrimidine dimers

  • 6-4 photoproduct

• ionizing radiation

  • ring opening

  • base fragmentation

  • breaks in covalent backbone of nucleic acids

• causes adjacent pyrimidine bases to bond with each other, forming dimers

  • instead of making H-bonds w/ something across → makes covalent bonds w/ itself

  • squishes 2 bases together + prevents info from being accessed

    • alters DNA structure + stalls replication → polymerase can’t read + falls off

nEta (translesion polymerase)

bypasses damage from cross-links

• no editing site

• not reading template

• larger active site fits distorted cross-linked Ts

  • adds 2 dAs to UV damage to synthesis strand

  • falls off → not processing

  • enables Pol III (prokaryotes) or Pol δ/ε (eukaryotes) access to 3’OH

    • can take over + replicate

  • damage still there

• usually adds dC if 8-oxo-guanine

alkylation damage

addition of alkyl groups to DNA bases

• from chemicals, eg. cigarette smoke, mold, burnt foods

• leads to mutations and disruption of DNA replication

• ex. O⁶-methylguanine → methyl added to gunanine carbonyl group

  • common + highly mutagenic lesion

  • methyl group blocks repulsive interaction b/w G + T oxygens

    • enables pairing w/ thymine rather than cytosine

DNA repair hypothesis

DNA = only biological molecule that life takes time + effort to repair

• DNA stability essential for encoding proteins/RNA

• DNA easily damaged

mutation

permanent change in NT sequence

• 1+ mutation → can become cancer

• types:

  • substitution = replacement of BP

  • insertion = addition of 1+ BP

  • deletion = deletion of 1+ BP

• caused by:

  • polymerase errors

  • DNA damage that then favours new BP interaction

silent mutation

mutation that affects nonessential DNA or has neglible effect on gene function

Ames test

test mutagenic potential of chemical compounds by observing if they cause mutations in DNA of bacteria

1. Salmonella typhimurium w/ mutation in histidine synthesis pathway grown on histidine-free plates

   1. something blocking histidine synthesis

2. Whatman filter paper soaked in potential mutagen

3. colonies arise in mutagenic conditions

   1. mutation relieves histidine synthesis blockage

DNA repair mechanisms

• base modification, abasic site

  • base excision repair (BER)

• crosslinks

  • nucleotide excision repair (NER)

• mismatches, insertions, deletions

  • mismatch repair (MMR)

• double strand breaks (DSBs)

  • homologous recombination (HR)

  • non-homologous end joining (NHEJ)

Dam methylase

functions in mismatch repair in E.coli - methylates GATC sites on adenine residues

• use single-carbon transfer co-factor SAM

• ~1min after replication, ______ methylates sites in newly synthesized daughter strand

mismatch repair (MMR)

fixes base-base mispairing + insertion/deletion

identification

1. Dam methylase recognizes GATC site on parent strand + methylates

   1. reads site in 5’→3’ direction

2. shortly after replication occurs → daughter strand doesn’t have methylation

   1. know which strand is parent + where mistake occurred

3. MutS + MutL loaded onto dsDNA at lesion (mismtach) using ATP

4. complex slides along DNA until GATC methylation site encountered

   1. can slide right or left → direction-independent

5. MutH cleaves non-methylated strand = single-strand nick made

repair

1. helicase unwinds DNA at nick site + moves to lesion

2. NTs b/w nick + lesion removed by exonuclease

   1. can be 5’→3’ or 3’→5’

3. nick or exonuclease generates free 3’OH

4. non-damaged strand used as repair template for DNA polymerase

5. gap at end of repair sealed w/ ligase

base excision repair (BER)

fixes modified bases + abasic sites

1. damaged base recognized by specific glycosylase → base removed = abasic site made

2. endonuclease cleaves phosphodiester backbone at basic site → 5’ phosphate nick

3. DNA pol I replaces missing base + does short extension via nick translation

4. gap filled by DNA ligase

DNA glycosylases

recognize common DNA lesions + remove affected bases by cleaving N-glycosyl bond in BER → abasic site made

• generally specific for 1 lesion type

  • eg. pocket inside specific for uracil

    • if uracil present in DNA → fit into pocket

    • senses it exists → shouldn’t be in DNA

    • hydrolyzes N-glycosidic bond

    • abasic site generated

nucleotide excision repair (NER)

repairs DNA lesions that cause large distortions in helical structure, ie. photo-crosslinks

1. lesion attracts excinuclease complex

   1. excinuclease: multisubunit enzyme that hydrolyzes 2 phosphodiester bonds on either side of distortion

2. proteins come along + recognize lesion

3. stay on strand + move along

4. make nick in backbone on either side of lesion

5. larger fragment stuck on template via base-stacking + H-bonds

6. helicase zips off fragment from template

   1. 3’OH on single-stranded template = substrate for DNA pol

7. DNA pol I (E.coli) or DNA pol ε (humans) fill gap

8. DNA ligase seals nick

MGMT (O⁶-methylguanine-DNA methyltransferase)

functions in direct repair → fixes alkylation damage

• catalyzes direct transfer of methyl group of O⁶-methylguanine to one of its own Cys residues

  • single methyl transfer event

    • Cys residue w/ thiol (SH) sidechain picks up methyl

  • permanently methylates protein → inactivated (suicide enzyme)

mechanism

1. binds to alkylated Guanine on minor groove side

2. binds w/ a-helical motifs, ie. Arginine

   1. arginine = (+) → interacts w/ (-) DNA backbone → stabilizes reaction

3. another arginine allows damaged base to flip out

4. base goes into active site where methyl group can be removed → covalently attached to Cys

double strand breaks (DSBs)

caused by:

• programmed DSBs

  • meiosis → recombination

  • V(D)J recombination

• incomplete NER or BER repair + subsequent DNA replication

  • polymerase gets to end of linear chromosome but doesn’t continue until end

  • blunt-end break at unrepaired site

• UV, IR or radiation damage

• replication stalling

  • polymerase hits cross-link

    • can’t read + falls off

    • no Etna to bypass → polymerase rejoins across damage

    • single-strand region in newly synthesized strand

repair foci

large macro-molecular bodies that form when cells are damaged by IR/DSB

• DNA repair proteins recruited and accumulate to facilitate repair

  • phosphorylation of H2A → yH2AX recruited to damage site

    • recruits protein 53BP1

      • binds to P53 → surveils genome

        • DSB detected = drives system towards apoptosis

        • no DSB detected = dissolves

    • PML-nuclear bodies dependent event → colocalize w/ persistent DNA damage foci

DSB repair pathways

1. HR

   1. template-driven

   2. conservative when sister chromosome used to repair break

2. NHEJ

   1. join 2 broken strands w/ microhomology

   2. can result in small deletions/insertions

nonhomologous end joining (NHEJ)

favoured pathway in humans (except in S phase → HR would be favoured)

• error prone → insertions + deletions if overhangs don’t align perfectly

mechanism

1. break recognition

   1. Ku70-Ku80 complex binds DNA ends + recruits repair factors

      1. help keep ends together to prevent strands from diffusing away

2. DNA-PKcs (kinase) bound to Artemis recruited

   1. phosphorylates → kinase cascade

3. Artemis “processes” site

   1. widens gap to allow more repair factors to bind

   2. has nuclease + helicase activity

4. broken DNA ends synapsed by Artemis

   1. on both sides of DSB, 1 strand of double helix lifted → flap

   2. overhangs anneal via antiparallel complementary WC interaction

5. Artemis removes single-strand extensions/hairpins → end-processing

6. XLF + XRCC4 + ligase IV complex seals up phosphodiester backbone

homologous recombination (HR) mechanism

1. nuclease resects DNA at 5’ end → large single-stranded DNA overhangs w/ 3’OH made

   1. resection = commitment to repair pathway

2. RPA binds to ssDNA overhang

   1. prevent strand from dipping into other DNA strands

3. BRCA2-mediated repair filament = traffic controller

   1. assembles repair filament that can go into intact DNA strand → invasion filament

4. 3’ overhang RPA displaced by RecA (E.coli) or RAD51 (humans) → becomes invasion filament

   1. controlled by BRCA2

5. BRCA1/BARD1-mediated strand invasion

   1. replaces BRCA2 + binds to RAD51 to form D-loop

   2. 3’ strand probes undamaged template for complementarity

      1. if NT complementary → RAD51 peels away like zipper

6. criss-cross formed = Holliday junction

   1. polymerase uses loop as template from free 3’OH as primer to fill in resected strands

7. second end capture

8. nuclease resolves Holliday junctions

9. ligase seals nicks

homologous recombination (HR)

high-fidelity repair pathway for DSBs in S or G2 phase

• 42% of human genome = line elements → repetitive

  • DNA broken in middle of line element → would go repair on any line element = ❌

    • translocation + fusion of 2 parts of chromosomes

• humans spend lots of time in G1 = long growth phase

  • do not have identical copies in G1

  • could end up in G1 if repair pathway used

• no insertions or deletions!

Holliday junction

structure formed during HR where 2 DNA strands exchange segments

• resolved by nucleases

  • nuclease makes single-strand nicks in backbone

    • why HR can be used to swap around genes during meiosis

• nicks sealed up by ligase

CRISPR-Cas9 mechanism

1. no RNA (CRISPR) = Cas9 protein in Apo (inactive) state

   1. PAM recognition region = disordered

2. Cas9 binds to CRISPR RNA → activated

   1. PAM recognition region = ordered

3. order PAM recognition region looks for PAM on target sequence

   1. PAM = 5’-XGG-3’ sequence

4. RNA molecule parts

   1. orange = structural component → binds to protein + fuels reorganization of PAM recognition site

   2. white = directs traffic

   3. purple = engineered sequence of interest → drives specificity

      1. complementary to NTs across from PAM on target strand

5. 1st purple NT makes antiparallel WC interaction w/ target strand → nucleation site

   1. continues to unwind → more WC interactions

6. R-loop made → RNA-DNA complex

7. after 15-20 NTs make WC interactions → large structural reorganization of HNH domain

   1. HNH moves up + over 3 NTs from PAM → active site opens + can cleave target strand

8. RuvC domain in open conformation

   1. lines up 3NTs away from PAM on non-target strand (w/ PAM)

9. blunt DSB generated 3 NTs away from PAM + complex falls apart

   1. RNA dissociates from DNA

Cas9

main protein in CRISPR

• 3 domains

  • PAM recognition domain

    • disordered = can’t recognize PAM

    • ordered = can recognize PAM

      • binds to lots of PAMs but falls off if no complemntarity found w/ target sequence

  • HNH domain

    • nuclease cleaving template strand 3 NTs away from PAM

  • RuvC domain

    • nuclease cleaving non-template strand 3 NTs away from PAM

CRISPR-Cas9

genome editing technology to generate site-specific break

• uses guide RNA + Cas9 protein to induce DSBs at specific locations in DNA

• DSBs repaired by endogenous repair pathways

  • NHEJ

    • can result in small insertion/deletion → gene disruption

  • HR

    • template added → HR driven

      • suppress proteins involved in NHEJ + drive HR

    • can add DNA sequence of interest