DNA Damage & Repair

Endogenous Damage

• from internal environment

DNA Damage

• chemical changes to the N-rich bases

• can lead to changes in base pairing potential → mutation

  • disrupts flow of info from that site in the genome

• can lead to double strand breaks → genome instability

• can be repaired or cells undergo programmed cell death

Spontaneous Deamination

• non-catalyzed (spontaneous) deamination of cytosine converts this base into uracil

• uracil chemical is very similar to thymidine → base pairs with adenine

• if left un-repaired, leads to A-T mutation

• non limited to cytosine

Deamination Reactions

• deamination = spontaneous loss of exocyclic amino groups

• deamination of cytosine to uracil = ~100 events/day

  • recognized as foreign (damage) in DNA and removed → why we don’t see uracil in the genome, and why DNA only contains thymine

Depurination Reactions

• depurination = hydrolysis of the N-β-glycosyl bond b/w the base and the pentose

• creates an AP (apurinic, apyrimidinic) site or abasic site; a site with no information

• more common with purine: double ring structure is a better leaving group than pyrimidines

• polymerase doesn’t know what H-bonds will be made

  • leads to mutations, replication stalling, double-stranded breaks

DNA Damage by Oxidative Damage

• reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radical) damage DNA

  • radicals really want to bind to something → if they bind to an N-rich base and add an oxygen, it changes how that base can interact w/ other nucleotides

• hydroxyl radicals are responsible for most oxidative DNA damage

• cells have an elaborate defense system to destroy reactive oxygen species

• as we get older, cells have more chances to accumulate reactive oxygen species

  • mitochondria leak more + release damaging oxygen molecules

DNA Damage by Oxidative Damage Figure

8-oxoG pairing

• 2 conformations of base and ribose (syn and anti)

• anti conformation is usually favored, but new steric clash b/w carbonyl and ribose oxygen now increases Hoogsteen base pairin b/w G and A during replication

• after a 2nd round of replication, this will generate an AT pair

8-oxoG and A base pairing

O⁶ Methylguanine DNA Methyltransferase

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

  • a single methyl transfer event permanently methylates the protein, inactivating

• damage does not lead to mutations until after replication

Exogenous Damage

• occurs from external environmental factors

• changes ability of N-rich bases to make canonical W-C interactions

• can lead to mutation after DNA replication

Pyrimidine Dimers promoted by UV Radiation

UV light causes pyrimidine dimers (2 thymines 2 cytosines in one row on one strand)

• cyclobutane pyrimidine dimers (2 covalent bonds) or 6-4 photoproduct (1 covalent bond)

• have the right geometry for covalent bonds b/w dimers when high energy radiation occurs in cell

• UV damage can cause photo-crosslinking (occurs b/w pyrimidine dimers)

• ionizing radiation (x-rays and gamma rays) causes: ring opening, base fragmentation, breaks in the covalent backbone of nucleic

Translesion Polymerase η Eta

• if the covalent bonds have formed, H-bonds b/w bases can’t form

• Eta is a translation polymerase that doesn’t repair UV-photo-cross linked DNA, it bypasses the lesion during replication by inserting the correct bases

• larger active site fits the distorted cross linked T’s

• no editing site

• is not reading the template (it’s not readbale)

• usually adds two dAs to UV damage, and usually adds a dC if it encounters 8-oxoG

  • then falls off → replication polymerase synthesizes the rest of DNA → prevents stalling

Foromation of Nucleotides w/ Alkylation Damage

• O⁶-methylguanine = a modified nucleotide that forms in the presence of alkylating agents

• common and highly mutagenic lesion

• tends to pair w/ thymine rather than cytosine

  • if left unrepaired, T will base pair w/ A in the next cycle (AT mutation)

• eg. cigarette smoke, moldy peanuts, burnt meat

Endogenous vs Exogenous Damage

Endogenous: spontaneous deamination, depurination, oxidative damage from respiration, DNA replication (polymerase errors)

Exogenous: photo-cross linking from UV (most common), chemical exposure (alkylation)

• both types of DNA damage leads to heritable mutations after replication

Mismatch Repair

DNA Polymerase Errors

• DNA polymerase has high fidelity: exonuclease site, shape discrimination, mismatch repair pathways

• some mutations sneak by from time to time, which lead to an error rate ~10^-9

  • mistakes can be catastrophic if it leads to certain mutations

• 3 billion base pairs: ~60 mistakes/cell division → mutation

DNA Methylation in E.coli

• cell can discern which is the parent strand and which is the daughter strand thru variations in DNA methylation

Dam Methylase (flagging system):

• methylates GATC sites on the adenine residues

• use the single-carbon transfer co-factor SAM

• ~1 min after replcation, this enzyme will methylate these sites in the newly synthesized daughter strand

• follows replication closely, parent strand is methylated, daughter strand is unmethylated

• this complex comes up and methylates the new daughter strand after replication

Post-Replication Hemimethylated DNA

Post-Replication Mismatch Identification

• non-canonical interactions distort the helix

• MutS and MutL are loaded on to the dsDNA at the lesion (mismatch) in an ATP dependent manner

• complex slides along the DNA until it encounters a GATC methylation site (GATC is used as a marker)

• non methylated strand is cleaved generating a single-strand nick (nuclease)

  • nucleotides are removed from the nick towards the damaged base

  • nuclease also requires a helicase to unwind the DNA

Post-Replication Mismatch Identification FIGURE

Mismatch Repair Steps

• helicase unwinds the DNA at the nick site and moves towards the lesion

• nucleotides b/w the nick and the lesion are removed by an exonuclease

• nick or exonuclease activity generates a free 3’OH

• non-damaged strand is used as a repair template for DNA polymerase

• gap at the end of the repair is sealed w/ a ligase

• dam methylase can methylate the GATC site

Mismatch Repair Steps FIGURE

Single-Strand Break Repair

• damaged DNA can be repaired in different ways, depending on the type of damage (binding site for repair proteins to recognize)

Base-Excision Repair (BER): Glycosylases

• DNA glycosylases recognize common DNA lesions and remove the affected base by cleaving the N-glycosyl bond in BER → generates an abasic site

  • generally specific for one lesion type

  • uracil DNA glycosylases recognize uracil in DNA and remove them

BER Steps

• damaged base is recognized by a specific glycosylase the removes the base (generates an abasic site which is a substrate for AP endonuclease)

• an endonuclease cleaves the phosphodiester backbone at the abasic site

• DNA Pol I replaces missing base (and short extension)

• Gap generated by the polymerase extension is filled with DNA ligase

Nucleotide Excision Repair in E.coli and humans

• occurs w/ larger distortions of DNA (e.g. photocrosslinking)

• exicnuclease: a multisubunit enzyme that hydrolyzes two phosphodiester bonds, one on either side of the distortions

  • distortion is a recognition site for exicnuclease complex

• a bunch of proteins bind to the lesion → spreads out in both directions → make 2 breaks in damaged DNA strand → allows helicase to come in and lift the whole damaged region out (flanking the damage site)

  • results in larger gap, filled w/ replicative polymerase (5’→3’)

    • DNA pol I (E.coli) or DNA pol ε (humans)

  • DNA ligase seals the nick

NER Figure

Direct Repair: MGMT

• requires even more specificity

• MGMT directly removes the damage (methyl-group) and the chemistry (binding potential) of the guanine residue is restored → restores to a canonical nucleotide

• MGMT is unable to release methyl-group after binding and degraded after a single reaction

  • single turnover protein (not an enzyme)

  • if we want to do the rxn again, more protein has to be synthesized

• costs the most ATP

MGMT

• binds to alklyated guanine residues – flips the base out and removes the methyl group – restoring the guanine to canonical W-C binding potential and mitigating mutation risks

• single turnover protein → costs a lot to make a protein

Direct Repair: MGMT FIGURE

Role of Cystein in MGMT

• Cys thiol group performs a nucleophilic attack on the alkyl group attached to the O⁶ position of guanine

• The alkyl group is transferred from guanine to cysteine

• irreversible reaction

Role of Arginine in MGMT

• helps with DNA binding and lesion stabilization

• Arginine is positively charged and DNA backbone is negatively charged

• Helps position the damaged guanine correctly in the active site

• May help flip the damaged base out of the helix (base flipping)

Cancer

• damage by exogenous or endogenous sources cause lesions in DNA, if unrepaired these can lead to mutations after DNA replication

• non-functional repair pathways can accelerate mutation, if these mutations disrupt the relationship b/w cell growth and division, this can lead to cancer

• carcinogens have high capacity to damage DNA and drive mtations and can be detected using Ames test

Mutations are Linked to Cancer

• mutation: a permanent change in the nucleotide sequence (in coding or non-coding regions)

• substitution mutation: replacement of one base pair w/ another (missense mutation); changes a.a in protein

• insertion mutation: the addition of 1+ base pairs; deletion mutation: the deletion of 1+ base pairs

  • caused by dsDNA repair pathways

• silent mutation: a mutation that affects nonessential DNA or has negligible effect on gene function

  • in coding region, doesn’t change a.a. in protein, but can change its ability to bind to tRNA

• nonsense mutation: a mutation tha causes a pre-mature stop codon (results in truncated protein)

• it doesn’t require many mutation in a cell in order to drive cell to transformation (uncontrollable growth, lacks cues for growth, cell division/cycle)

  • takes very specific mutation

Ames Test

• detects mutagenic compounds

• Salmonella thpimurium w/ a mutation in the His synthesis pathway is grown on His-free plates (can’t make His)

• a small circle of Whatman filter paper is soaked in a potential mutagen (ask if it drives mutations)

• colonies will arise in mutagenic conditions, after the mutation relieves the histidine synthesis blockage

• the further we get colonies, the more mutagenic (even at dilute conc’s we’re getting mutations)

Ames Test FIGURE

DNA Repair is Tumor Protective

• cancer is driven by changes in genome of a cell that allows it to be oncogenic (often involves mutations in DNA repair genes)

• DNA damage can be used as a cancer treatment: damages DNA at tumor site, if you get enough damage, it can drive cell to apoptosis