DNA Repair

What are the spontaneous DNA mutations that require DNA repair?

1. Depurination

2. Deamination

3. spontaneous oxidative destruction

4. uncontrolled methylation

5. hydrolytic attack

6. UV expose (non-spontaneous I think)

7. Double stranded DNA breaks

What is depurination?

Spontaneous loss of ~5,000 purines (A or G) bases lost per cell per day because of hydrolysis of the glycosyl linkage to deoxyribose 

• Occurs when the bond connecting the purine to the sugar is broken by a water molecule 

  • Resulting in a purine free nucleotide that can’t act as a temple during replication 

  • A hydroxyl group is left instead of the nitrogenous base group 

• MOST COMMON 

• Can result in a complete loss of both bases (deletion of base pairs) 

What is deamination?

Spontaneous removal of the amine (NH2) group from ~100 cytosines per cell per day to form uracil 

• Occurs when hydrolysis wipes away the NH2 from a cytosine and replaces it with an oxygen. Forming an uracil and an ammonium ion 

  • C => U 

• Changes the base pairing  

What is spontaneous oxidative destruction?

Reactive oxygen radicals or chemicals exposure cause base damage 

• Red arrows 

What does the green arrows represent

Uncontrolled methylation

What do the blue arrows represent

Hydrolytic attack

What is UV exposure (non-sponantous)?

Forms pyrimidine dimers via covalent linkage between two bases 

• A covalent bond is made between 2 bases 

Pyrimidine dimers = can block transcription/replication when brought close together and not repaired 

What is double stranded DNA breaks

occurs frequently with ionizing radiation issues, replication errors, & oxidizing agent destruction 

What is BER (Base Excision Repair)?

removed affected base and replacing it with the correct one 

• DNA glycosylases are specific for altered DNA base (DETECTION) 

  • DNA glycosylase enzyme flips out each nucleotide until it finds one to repair 

• Steps (REPAIR PROCESS): 

1. Hydrolysis by specific DNA glycosylase 

   • Flips out nucleotide 

2. AP enzymes (aqurinic or apyrimidic endonucleases) chew out sugar backbone here 

3. DNA polymerase adds new nucleotide 

4. DNA ligase seals nick with new sugar piece 

What is NER (Nucleotide Excision Repair)?

Removing a ~30 nucleotide chunk of affected strand 

• A protein recognize a dimer begins this repair (DETECTION) 

  • The errors produce a atypical “bulky lesion” (not normal for DNA helix 

    • Large multienzyme complexes see the lesion and cleave out the phosphodiester backbone  

    • DNA pol. Helicase, and ligase go to single strand to copy over 

• Steps (REPAIR PROCESS): 

1. Helicase unwinds local duplex 

2. Excision nuclease cleaves on both sides of damage leaving a gap of ~30 nucleotides 

3. DNA polymerase and ligase finish job 

What is transcription-coupled repair?

Errors repaired once transcription has started (past replication) 

• RNA polymerase will stall transcription where it finds a DNA error 

  • It'll reverses itself and call over coupling proteins 

  • Coupling proteins will repair the error (direct excision repair machinery (calls over NER)) and leave 

  • RNA polymerase continues transcribing 

What is NHEJ (Non-homologous end joining)

Repairs double stranded DNA breaks by removing a set of nucleotides, then ligating the strands back together (does delete part of strand) 

• DNA polymerase µ & λ (family X) involved  

Negative side effect = often results in deletion mutations or translocations 

• Acceptable because majority of genome is non-gene coding

What is HR (homologous recombination)?

repairs double stranded DNA breaks 

• A more accurate mechanism (than NHEJ) 

• Occurs only AFTER replication (S and G2 phase) has occurred, when there is a nearby duplex daughter DNA to serve as the template 

Negative side effects = 

• Loss of heterozygosity 

• Possession of these proteins is often controlled by other proteins like BRAC1/2  

  • These alterations can cause cancers 

How does DNA base chemistry facilitates DNA damage detection?

What is XP(Xeroderma Pigmentosum)? phenotype, process affected, and mutation

Thymine dimers (made via UV damage) block transcription and replication because the repair enzymes are mutated 

• Dysfunctional repair enzyme that is HERITABLE (germ line mutation) 

  • Can result in tumors  

Phenotype: Skin cancer, UV sensitivity, and neurological abnormalities 

• Non-ionizing radiation forms the dimers and there isn’t a repair enzyme available to fix them 

Process affected: NER (dysfunctional repair enzyme) 

What is Cockayne Syndrome? phenotype, process affected, and mutation

Defect in transcription-coupled repair 

Mutation in 2 genes on chromosomes 5 & 10 

Phenotype: UV sensitivity, developmental abnormalities (underdeveloped tissues/organs) 

Process affected: coupling of NER to transcription 

• Because there is so many lesion sites (errors) RNA polymerase gets stalled and there is insufficient genes (certain genes) 

  • Lesions are from UV damage 

What is AT? phenotype, process affected, and mutation

Single nucleotide base change (point mutation (via deletion, insertion, or substitution)) 

Phenotype: Leukemia, lymphoma, genome instability, and gamma-ray sensitivity 

Process affected: ATM protein (a protein kinase activated by double-strand DNA breaks 

What is BRCA1? phenotype, process affected, and mutation

Spontaneous mutation on brca1 gene 

Phenotype: Breast and ovarian cancer 

Process affected: Repair by homologous recombination 

What is the consequence of a loss of DNA repair gene

Unrepaired spontaneous errors can become permanent if they don’t get fixed 

• DNA repairs depends on healthy DNA repair genes to make functional repair enzymes/proteins 

• Many repairs in 24hrs (more than 1,000 for some) 

Semi-conservative replication/replication allows the “good” strand can be used to restore damage or continue functioning as normal 

What are the DNA polymerases involved in DNA repair?

Family B (α, ε, & δ) = Replication 

Family X (β, µ, & λ) = BER/NER repair  

Family Y (n, l, & k) = translesion polymerases 

• Don't have exonucleolytic activity 

• Don't discriminate about which nucleotide to add 

• Often leads to errors 

• Come and rescue Family B polymerases when they are stuck trying to replicate 

• Once pushes through error, will leave strand so DNA polymerase family B can continue