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Straßen restlichen A (Fach) / dfgdfg (Lektion)

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• 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.  
• 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. 
• 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
• 45. Briefly depict the process of homology directed repair with synthesis dependent strand annealing (no protein names are necessary, but give the neccessary terms for the DNA structures) Single-stranded DNA  generated at break sites by helicases and exonucleases • Synapsis: One of the ends pairs with an intact duplex by invasion, generating a heteroduplex and forming a displacement-loop D-loop • Once the D-loop forms, repair can proceed in several different ways, including synthesis-dependent strand annealing (SDSA) ·         DNA synthesis continues until the new DNA can pair with the other (gray) 3’ overhang ·         • Replication proceeds as repair and then ligation finishes the process
• 53. Where is RuvAB involved in the broad complex of recombination? Bacterial tetrameric RuvA holds the junction in an open conformation and recruits RuvB ATP dependent helicase. This breaks and re-forms base pairings to move the Holliday junction.
• 56. How can damaged replication forks (specifically nicks) be repaired with a principle reminiscent of recombination? single strand nick leads to a ds break after strand separation. One strand of the "broken off" end invades the "whole" part a D-loop structure is formed, DNA synthesis occurs, the displaces strand is captured by the lagging strand lagging strand synthesis is restarted and after clevaged the fork is restored see
• 59. Name at least two problems that can occur with recombination due to sequence similarity regions? Recombination on the same chromosome Unequal crossover between chromatids or between homologs can lead to deletions and duplication Crossing over between different chromosomes can lead to DNA translocation
• 66. Does transposition necessarily require homology? No it doesn´t. Insertions can result in target-site duplications - small sequences at the insertion site that become duplicated due to DNA repair mechanisms. 
• 75. How is it ensured that transposon ends are brought together before cutting for cut and paste DNA elements? The transposase cuts in trans, i.e. they cut the other end that they don't bind.
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• 78. After a DNA cut and paste transposon leaves a certain site a precise excision can occur. Explain what this is and name conditions. When the transposon leaves and the resulting gap can be repaired by homology directed repair from a transposon free site, the original state can be restored and no footprint remains. 
• 79. Explain in broad, superficial terms where VDJ recombination occurs? Antibody heavy chains are encoded by four gene segments –there are many versions of each V(ariable), D(iversity) and J(oining) segments and a C-terminal invariant region (encoded by the C(onstant) locus). 
• 80. What is the relationship between VDJ recombination and transposons? They are related:   o   Lymphocyte-specific recombinases are related to transposases o   Related in the signal DNA sequence
• 82. Name an application in the lab for the Drosophila P element? Using microinjection this is a way to make transgenic flies.
• 85. Within a LTR Retroelement you usually find coding regions for 4 proteins, name these? INtegrase, GAG, PRotease, RT-RnaseH reverse transcriptase+ RNAse activity
• 87. Describe how a LTR element multiplies and integrates into new regions? • LTR elements are transcribed into a provirus by host machinery and exported from the nucleus • Proteins are produced from the mRNAs and the RNAs and proteins assemble into a virus-like particle • In the virus-like particle, RNA is converted to dsDNA by reverse transcription • The DNA is integrated into the host by integrase • In retroviruses there is an extracellular stage that requires ENV
• 88. What can happen when you have two LTR elements spaced a bit apart from each other with the intervening DNA? It can get lost due to homologous recombination between the elements.
• 93. What is the sequence requirement for an insertion of some non-LTR elements (e.g. LINEs) and why? a stretch of Ts that pair with the polyA tail
• 94. How does DNA methylation affect IS10 transposition? DNA methylation both prevents transposase expression and transposon end activity
• 96. What is the role of piRNA? They block transcription of certain transposons.
• 97. Ty5 is a relatively "benign" element when it comes to gene mutations why? It preferably inserts into heterochromatin
• 98. Explain what local hopping means for certain transposons? If transposons preferably insert in a location close to the donor site.
• 102. What is the difference between Serine and Tyrosine Recombinases when it comes to the intermediate form, describe both forms? Tyrosine recombinases break DNA and form DNA-3P-tyrosine linkage, Serine recombinases break DNA and form a DNA 5P-serine linkage.
• 103. Draw and explain the recombination of a serine resolvase. ff
• 104. Draw and explain how the Cre recombination occurs (a tyrosine recombinase). The bateriophage P1 Cre-lox System ·         Recombination can occur at Cre-lox sites • The homologous spacers (shaded boxes) have directionality (green arrow) • The spacer is surrounded by recombinase binding sites (blue and pink arrows) • Four recombinases bind to two duplexes • DNA is cut in a staggered manner so the recombined DNA molecules contain heteroduplex spacers         • The requirement to form heteroduplexes enforces the accuracy of the recombination
• 105. What is the Cre enzyme and what does it do? a (tyrosine) recombinase that recognizes lox sites.
• 106. For what can the Cre/lox system be used for? To delete DNA between two lox sites, this is often used as a tool where Cre is expressed under a specfic promoter. After Cre expression, a piece of DNA is removed and for example a tissue specific knockout is made.
• 108. What is the Invitrogen Gateway system system building on (which molecular mechanism from which organisms)? phage lambda recombination

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