ISALAN - dna repair Flashcards
Eukaryotic genome
- highly organized in a hierarchical order:
o consist of double helix DNA wrapped around histones
o further form higher order structures by supercoiling
o eventually form a condensed chromosome
Bacterial genome
- genomes vary – some linear, some circular
- less ordered than eukaryotic, but still highly organized
o Still have coiling and generalized protein interactions that scaffold it & protect it from damage
o no histones, but histonelike + charged proteins that interact with DNA ex. H-NS:
Histone-like nucleoid structuring protein, Fis protein
DNA synthesis
- by DNA polymerases in 5’-3’ direction because it facilitates the 3’OH nucleophilic attack on the incoming NTP
- Require hydrolysis of ATP, Mg2+, nucleoside triphosphates
DNA damage
- DNA is damaged approximately 10,000 times per cell per day by environmental mutagens (eg. smoke, chemicals, radiation, UV light, pyrolysed food)
- DNA damage can: block replication and/or transcription; cause alterations in the genetic code
Sources of DNA damage
- Exogenous source (outside cell): environmental mutants ie. UV radiation, benzo[a]pyrene in burnt food
- Endogenous source (inside cell): from internally generated damaging agents which are by-products of natural metabolism, such as hydroxyl radicals
- Spontaneous damage: even if DNA is protected from external chemicals & radiation, spontaneous damage will still occur within the DNA itself (naturally occurring damage)
Exogenous source
- UV light induces formation of pyrimidine dimers (most common = thymidine dimers), in which 2 adjacent pyrimidines are joined by a cyclobutene ring structure (a strong covalent lesion which interferes with base-pairing during replication, causing mutations in the genetic code)
> Explains why solar UV radiation is the cause of most human skin cancer - Carcinogens react with DNA bases, resulting in the addition of large bulky chemical groups to the DNA molecule
> These bulky groups can:
1. Prevent DNA/RNA polymerase from properly moving through DNA
2. Disrupt Watson-crick base pairing potential, causing mutations during DNA replication
Endogenous source
- Incorrect methylation of DNA (non-indigenous methylation of bases) by internal agents changes the Watson-crick base-pairing potential of the nucleotides by changing the number of H bond donors/ acceptors
Eg. methylation at O6 of Guanine – disrupt the C=O bond that is a H bond donor
involved in the base pairing of Guanine (changes H bond properties with Cytosine)
Spontaneous damage
- Deamination: removal of amine from DNA bases (namely adenine, cytosine, guanine) replacing it with =O, which will alter the DNA base type and its base pairing potential
Eg. deamination of Adenine to Hypoxanthine disrupts AT base-pairing potential, resulting in an AC pair instead (hypoxanthine looks more similar to guanine which is able to form 3 H bond with C)
> because of deamination of A, DNA is receiving incorrect information during replication (AC pair instead of AT), creating a permanent mutation in the DNA which can be transferred down generations - Depurination: results from cleavage of the bond between purine base and deoxyribose sugar, leaving an apurinic (AP) site in DNA (gets rid of an entire purine)
> This results in complete loss of bp potential & loss of genetic information from the cell
DNA repair mechanisms
2 general types of DNA repair mechanisms
- Direct reversal: the chemical reaction responsible for DNA damage is reversed back to the original form
> More specialized kind of repair that varies between organisms since it involves
specific enzymes for specific DNA damages
- Excision repair: removal of the damaged bases and replacement with newly synthesized DNA (can be single bases or stretches of DNA)
> More common & most important in humans
Direct reversal (examples)
- Photoreactivation – direct reversal of pyrimidine dimerization caused by UV exposure
* Thymidine dimer causes a lesion in DNA backbone – recognized by Photolyase
* Photolyase is a photoreactivating enzyme that uses visible light to break the cyclobutene ring to free up the pyrimidines
* Occurs in E. coli, yeasts, some plant and animal cells but NOT in humans (has another way of reversing thymine dimer formation) - O6-methylguanine methyltransferase (widespread in prokaryotes and eukaryotes) demethylation/ reversal of the non-indigenous methylation of O6-methylguanine
* Utilizes cysteine in the active site which is able to oxidize and form bonds with the methyl group, removing it from O6-methylguanine and form methylcysteine in the active site instead
* The cell now has to generate energy/ reducing power to reduce methylcysteine back to reactive cysteine, so the enzyme can be reused
Excision repair
3 types (based on size & when they occur):
- Base-excision repair: a single base is removed, leaving the deoxyribose backbone intact
- Nucleotide-excision repair: a whole region of nucleotides (several bases ) are removed, resulting in a gap in 1 strand
- Mismatch repair (post replicative repair): happens after DNA synthesis. It is a repair mechanism specific for damage occurred due to error of DNA polymerase
Base excision repair
Starts off with DNA containing a lesion/mismatch so they are not H bond correctly (ex. uracil formed by deamination of cytosine, leads to a GU mismatch)
1. uracil DNA glycosylase recognizes the lesion caused by GU mismatch
2. uracil DNA glycosylase cleaves the bond between uracil and the
deoxyribose sugar, leaving a sugar with no base attached in the DNA (an AP site). This results in loss of info, and further process for info restoration is required.
3. AP site is recognized by AP endonuclease, which cleaves the DNA chain (makes a nick inside the DNA by cleaving phosphodiester backbone). The remaining deoxyribose is removed by deoxyribosephosphodiesterase.
4. The resulting gap is now suitable for the normal process of DNA synthesis so it can be filled by DNA polymerase and sealed by ligase - leads to correct incorporation of C opposite G.
Nucleotide excision repair (general)
Ex. repair of thymidine dimers
1. The thymidine dimer causes a lesion in the DNA
2. Damaged DNA is recognized by 3’ and 5’ endonucleases which creates nicks on both sides of the thymine dimer
3. Helicase unwinds the DNA, resulting in the excision of the oligonucleotide segment containing the damaged bases
4. The resulting gap is then filled by DNA polymerase in 5’ to 3’ direction and sealed by ligase, recovering the DNA chain
- The precise components involved in the mechanism varies between organisms:
Nucleotide excision repair (E. coli)
Catalyzed by 3 gene products: UvrA,B,C
1. UvrA recognises damaged DNA
2. UvrB and UvrC are endonucleases that cleave DNA at 3’ and 5’ sides and contain
helicase activity that excise the 12-13 bases oligonucleotide segment
3. Mutations of these genes leads to high sensitivity to UV
* DNA pol 1 is responsible for filling in gap after removal of oligonucleotide segment
Nucleotide excision repair (humans)
- Catalyzed by RAD gene products which involve 7 highly conserved repaired genes
(involve more components but similar machinery to E. coli)
1. identified by studying xeroderma pigmentosum, a rare genetic disorder affecting 1:250,000 people
2. affected individuals show damage in the 7 repair genes, thus are deficient in nucleotide-excision repair ability, resulting in an extreme sensitivity to UV light.
3. This means that certain DNA damages can’t be removed from the human cell quickly, so they accumulate (acute damages can be healed but the disease can lead to skin cancer, so overall, it affects the organism’s viability and capability to survive & reproduce) - In humans, DNA pol β is responsible for filling in the gap after removal of oligonucleotide segment
