DNA replication mechanism and damge repair Flashcards
telomerase
gap of end of lagging strand where RNA primer gets excised
-telomerase extends the template, makes another RNA primer and DNA to fill the gap so the entire template can be replicated
DNA pol proofreading
- a 3’ to 5’ endonuclease to remove wrong nucleotides
- polymerase has active sites for polymerization and for exonuclease
- mismatches do not have high fidelity for polymerase active site, giving it time to move to exonuclease site
DNA replication limited to one per cell cycle
- pre replication complex
- ORC binds chromatin at M or G1 phase, Cdc6, Cdt1, then MCM2-7 is DNA licensing
- 22 more proteins + cyclin regulation before replication will begin
replication stress induced genome instability
drugs or mutations cause stress
- deregulation of origin of regulation: deregulated CDKs, inhibiting CDKs, nucleotide shortage may all induce stress
- replication stress = rep fork stalling
- activates checkpoint pathway which includes ATRs and RAD proteins
- stops replication to protect fork from breaking or gaps - impediments: centromers, telomers, repeats, RNA, lesions, all trigger the above checkpoint pathway
- transcription/replication may interfere bc they use the same template. long strands being transcribed take more than 1 cell cycle. could run into replication bubble and cause DNA to break
types of DNA damage
- spontaneous - all macromolecules subject to decay
2. mutagens - in environment; damage DNA and cause mutation
endogenous DNA damaging reactions
- depurination: cleave glycosidic bond at deoxyribose/purine base
- deamination: cytosol-> uracil
- oxidation: guanine-> oxoguanine, thymine-> thymine glycol
- nonenzymatic methylation: by S adenosylmethionine
- break strand
environmental mutagens causing DNA damage
- ionizing radiation - X rays
- DNA methylating agents: MNNG
- Bulky hydrocarbons- benzypyrene in cig smoke
- DNA groove binders - distorts helix
- DNA intercalators: inserts aromatic mlc between base pairs to unwind, prevent replication/transcription
- DNA cross linking: forms N bonds, prevent rep and transcription and cause cell death. used in anti cancer
- UV cross linking - cyclobutane joing 2 thymes to block replication
* if not repaired, no transcription –> cell death
DNA repair mechanisms
direct DNA repair: 1. Photoreactivation 2. methyl guanine methyltransferase single strand: 3. Base excision repair 3. nucleotide excision repair 4. ribonucleotide excision repair 5. mismatch repair
double strand:
- non homologous end joining
- homologous recombination
photoreactivation
visible light by photolyase repairs dimers
MGMT
- takes alkyl group off of guanine
- MGMT gets alkylated and turns gene on to make more MGMT to repair
- if gene gets methylated, MGMT is downregulated; this methylation may be carcinogenic
Base excision repair
- glycosylate removes damaged base pair
- exonuclease cleaves bond
- replace with undamged base pair and seal
- linked to cancer and neurological disorders
Nucleotide excision repair
- xeroderma pigmentosum (XP) disease
- RPA and XPA recognize damge, make bubble and assemble complex
- endonucleases cleave on BOTH sides of damage
- repair with polymerase and ligase
mismatch repair
- repairs 99% of errors
- would lead to permanent mutation in next gen if not
- inheriting a mutation in one of the mismatch repair genes inc chance of cancers like Hereditary non polyposis colon cancer
- starts with one allele mutation but since they lack repair enzymes second is more likely to mutate as well = mutator phenotype
Ribonucleotide excision repair
-RNA added instead of DNA
-RNaseH2 repairs it
Aicardi Gourtieres syndrome: RNase H2 deficiency
non- homologous end joining
DNA ligase joins two broken ends
