Apricio - Lecture 7-9 Flashcards
Isomerization of Bonds in Bases –> alter base paring capabilities
- predominant form: (amino for Cytosine), (keto for Guanine)
- altered form is frequent enough to contribute to errors during DNA replication that leads to a mutation (if not corrected)
Mutation frequently is caused by…
base mis-incorporation during DNA replication; not yet a full mutation since it is detected and repaired usually; DNA replication before repair (1/10E9) will stabilize the mutation in DNA
Means to recognize newly synthesized strand - E. coli (Hemi Methylated DNA)
5’-GATC is methylated - DNA methylase
nascent DNA (for certain period of time) is not methylated
==> nascent and original DNA strands can be distinguished–>can keep original information
Mismatch repair (Non-humans) *MutS, MutL, MutH, ExoVII or RecJ (5'->3'), ExoVI(3'->5'), helicase II, ATP, DNA polymerase III, holoenzyme, SSB, ligase*
- MutS - Scans DNA & detects bulges in backbone (resulting from mispairing)
- MutL&MutH binds to hemi-methylated DNA; identify nascent(unmethylated) DNA
- MutH endonuclease cleave unmethylated DNA strand (nick)
- exonuclease remove a stretch of DNA containing mismatch
- DNA polymerase fills in the gap
- Ligase seals the nick
Different types of Exonulcease in mismatch repair
Positions of methylated DNA and MutS are important
- 5’ to 3’ : ExoVII or RecJ
- 3’ to 5’ ; ExoVI
Replication slippage - at short, tandemly repeated sequences
*neurological disease related to this mutation
- hairpin (on nascent) == without repair-> mutant DNA will have longer DNA sequence (extra repeats)
- hairpin (on template; less common) == without repair -> deletion of few repeats on synthesized strand
DNA base damage
- deamination of cytosine -> uracil => recognized as foreign
- depurination/depyrimidation -> entire loss of a base => leaves an abasic/unpaired site *DNA backbone is intact ==> block replication
- deamination of 5-methyl-cytosine -> thymine => not obviously foreign
- Various modifications of guanine; e.g. 6-methyl-G pairs with T, 8-oxo-G pairs with A
Deamination of 5-methyl-cytosine
(eukaryotes) cytosine followed by guanine often methylated
deamination of 5-methyl-cytosine => thymine
if not repaired. T will pair with A creating stable mutation
Radiation and DNA damage
UV radiation: double bonds in pyrimidine rings activated to react with adjacent base; crosslink between pyrimidine bases (e.g. thymine dimer; cannot be read, stops DAN polymerase)
2. Ionizing radiation (gamma-, X-ray): create dsDNA breaks, cause dleletion/insertion/chromosomal rearrangements
Ames Test
Potentially mutagenic compound screened quickly in bacteria (e.g. salmonella)
reversions (reverse mutations) of mutations can be tested easily
Mutagenic
compounds that are not mutagenic -> converted to mutagens in the liver
-> liver enzyme treating can test this possibility
Photoreactivation
T-T sequence + UV radiation -> crosslink; pyrimidine dimer; thymine dimer -> DNA photolyase attaches to the mutation site (Dark) -> visible light activates/catalyzes the reaction
Base excision repair
removal of uracil/damaged base
- DNA glycosylase cleaves base
- abasic nucleotide removed by AP endonuclease and exonucelase
- gap filled with DNA polymerase I and ligase
Nucleotide Excision Repair
Fix: Damaged bases, pyrimidine dimers (crosslink), as well as larger adducts
- UvrA/UvrB: scan distortion on DNA
- UvrB: opens the helix by bending. recruits UvrC
- UvrC (endonuclease); cleave ssDNA on both ends of the lesion
- UvrD helicase: remove nicked NDA
- DNA Pol I & Ligase
- defects in this pathway causes = Xeroderma pigmentosa
Translesion DNA synthesis (Pol IV, PoV)
- special (translesion) polymerase that can synthesize past the lesion without base pairing
- no template used; highly error-prone
- non-processive
- used only when necessary; result of SOS response in bacteria or DNA checkpoint response in eukaryotes
- can recognize error in ssDNA
- other methods work on dsDNA
