DNA repair Flashcards
Give examples of heritable human diseases that are caused by defective DNA repair pathways.
- Xeroderma pigmentosum: caused by mutation in global nucleotide excision repair pathways.
- Cockayne syndrome: caused by mutations in transcription-coupled nucleotide excision repair pathways.
- Lynch Syndrome: caused by mutation in Mismatch repair pathway
Describe the sources and nature of damage to DNA, the type of machinery used to repair the damage, and the molecular consequences of failure in DNA repair (8 types of damage)
- Spontaneous hydrolysis (deamination, depurination). Repaired by BER
- Deamination: can lead cytosine to become uracil, which may be mis-incorporated into DNA (point mutations). Repaired by BER
- depurination
- alkylation→ point mutations
- oxidation via ROS→ blocking DNA replication or point mutations
- UV radiation→ formation of pyrimidine dimers. Repaired by direct reversal or NER
- Adduct formation→ intercalating agents creates adducts in DNA, interfere with DNA replication. Repaired by NER
- DNA strand breaks (double-strand breaks): interfere with DNA replication. Repaired by strand break repair or direct reversal
Explain the basic steps of mismatch repair, describing the type of damage repaired by this pathway, and understand the marking of the old strand of DNA by methylation in E. coli
- Corrects for mistakes made by DNA Pol during replication
- Step 1: MSH and MLH (MutS and MutL in bacteria) bind DNA. MSH and MLH are mutases.
Step 2: MSH recognizes misincorporated nucleotide and MLH forms nick (signals DNA helicase and exonuclease to come in). excises the mismatched base from the daughter strand (parent strand marked via methylation)
Step 3: gap repaired by DNA Pol and ligase
Describe the basic mechanisms of base excision repair
BER
Step 1: modified base recognized by DNA glycosylase, which removes the base from the sugar to form an AP site
Step 2: AP site-specific endonuclease cuts phosphate backbone 5’ to AP site
Step 3: Another endonuclease cuts 3’ of AP site
Step 4: gaps is filled by DNA polymerase and DNA ligase
Describe the basic mechanisms of nucleotide excision repair
NER
Step 1: global or transcription-coupled recognition of damage
Global recognition involves recognizing damage anywhere in genome; different proteins used
transcription-coupled recognition involves different set of proteins
Step 2: TFIIH complex binds to DNA, and helicase portion of complex unwinds DNA to form bubble
Step 3: endonucleases cut into DNA and remove 30 bp segment containing lesion
Step 4: gap is filled in by DNA Pol and rejoined with DNA ligase
Describe the basic mechanisms of Double Strand Break Repair
Fixed by either homologous recombination (need sequence homology), or nonhomologous end-joining (non homology needed)
Describe the mechanism that enables replication to continue in the face of DNA lesions that other repair pathways fail to remove, and know the unfortunate consequence of this process for the cell.
DNA damage tolerance/bypass:
- DNA acquires too much damage for cell to handle
- Cell bypasses DNA repair pathways using a “looser” DNA polymerase to continue replication. These DNA Pols lacks exonuclease activity meaning it doesn’t proofread the replicated DNA.
- DNA Pol η extends new strand over lesion, while DNA Pol ι extends new strand beyond lesion.
- Lack of proofreading leads to mutations which could lead to cancer.
Explain the concept of DNA damage checkpoint and its role in maintaining genome stability.
- DNA damage checkpoints pause the cell cycle to allow DNA repair before replication
- Recognition of DNA damage leads to kinase activity, that leads to arrest of cell cycle.
- Stalled replication and DNA lesions activate checkpoint kinases
- ATR and ATM are activated, after which point they phosphorylate Chk1/2
- Chk1/2 then activate other proteins, leading to DNA repair, arrest of cell cycle, or activate of p53 and permanent cell-cycle arrest.
