Molekularbiologie (Fach) / repair (Lektion)
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- 29. Was ist transition und transversion? Transition: Purin vs. Purin, Pyrimidin vs. Pyrimidin Tranversion: Purin vs. Pyrimidin, vice versa
- 30. Name some sources which can lead to mutation? · Radiation (UV) · Internal cell metabolites · Tautomeric structures of DNa bases · External chemicals · Deamination
- 31. What is a simple mechanism to repair UV induced tymine dimerization? (direct reversal) Direct reversal of the damage by photolyases. For example they reverse pyrimidin dimerization.
- 32. Alkylation repair. What is special about it? (direct reversal) Alkylation on guanine or the DNA backbone is repaired by alkyltransferase. Once bound to an alkyl group Ada is inactivated. Methyl Ada stimulates more Ada production and also stimulates AlkA production (another repair protein). Ada transfers a methyl group from the damaged alkylated DNA to its active sites. Methylated Ada activates expression of its own gene and a glycosylase gene.
- 33. Mismatch repair? Mis match repair fixes mistakes made during replication, using the other strand as a template. The mismatch is recognized by MutS protein the new strand by MutH (new DNA is not methylated). MutSLH complex assembles and MutH nicks the new strand. The nicked strand is digested by an exonuclease and DNA is resynthesized. Mismatch repair can detect and repair hairpins – the newly synthesized DNA is degraded, the hairpin unfolds and the new strand can be re-made. Defects in mismatch repair lead to increased rates of cancer and spontaneous mutation (this is a mutator phenotype).
- 34. Base Excision repair? When it is used? Damaged bases are removed by glycosylases. AP endonucleases cleave the backbone and the gap is filled by repair polymerases. AP endouncleases also become active if the base has been lost for any other reason.
- 35. Nucleotide excision repair. It is used for bulky lesions, which distort the DNA helix and 10-30 bases are excised and repaced. In bacteria, UvrA and UvrB scan DNA for distorted regions, UvrB (a helicase) unwinds the damaged region. UvrC is recruited and nicks the damaged DNA and damaged DNA is then removed by UvrD. DNA is resynthesized from the undamaged strand.
- 36. TLS Polymerase? Damaged DNA may fail to be repaired before it meets a replication fork, and can lead to stalled replication forks. After replication forks stall on damaged DNA, translesion polymerases (TLS) can take over synthesis. TLS polymerases can work in a non-template fashion. After TLS polymerases finish, the normal polymerases take over once again. Because of their low fidelity, TLS polymerases must only be recruited when necessary. TLS polymerase active sites are more open and flexible than replicative polymerases. This flexibility allows then to bypass problematic DNA and continue synthesis, albeit with lower fidelity.
- 37. Why are TLS polymerase a last resort? The TLs polymerase are error prone and just ensure that DNa can be replicated, this often leads to mutations. It can safeguard replication though.
- 38. LEXA/RECA system. · damage response · replication fork stallsà single-stranded DNA is exposed – RecA binds · Bound RecA forms a filament on single-stranded DNA, and stimulates cleavage of LexA. Cleaved LexA can’t bind DNA, SOS genes are transcribed. SOS repair proteins fix the damaged DNA. RecA activity decreases, and LexA rebinds DNA to turn off SOS gene transcription. LexA also inhibits cell cycle progression, to gain time for damage repair.
- 39. Where do ATM and ATR play a role? They play a role in single and double stranded DNA breaks in eukatiots. Sensors recruit the Regulator kinases (ATM and ATR), which mediate the damage response.
- 40. Effects of atr activation? o Cell cycle control o Replication fork stabilization o Replication origin control
- 41. Explain the regulation of p53. Apoptosis is orchestrated by p53. p53 acts by binding at promoters of target genes to increase proteins that inhibit the cell cycle or stimulate apoptosis, and decrease apoptosis inhibitor. p53 activation is designed to cause cell death, so its activity is carefully regulated. This is done by posttranscriptional modification (phosphorylation, acertylation,…) · When no DNA damage is present MDM2 ubiquitinates lysines in the p53 C-terminal domain, targeting it for degradation. Any remaining p53 is in any case exported from the nucleus · Kinase activity stimulated by DNA damage results in phosphorylation of p53 and MDM2, so they no longer interact · p53 tetramerizes, blocking nuclear export · p53 interacts with transcription proteins, including p300, which acetylates histones (enhancing transcription) and p53