Molekularbiologie (Fach) / recombination (Lektion)
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- 42. What is NHEJ? Non-homologous end joining. It simply rejoins the ends. It is the main mode of repair of double stranded breaks in mammalian cells. Predominant in non-dividing cells.
- 43. Simplest mode of NHEJ? A double stranded break is repaired by simply joining the ends potentially after trimming a few bases by a nuclease.
- 44. Resection during NHEJ! What it that? o Resection to expose single stranded DNA in NHEJ o Base pairing occurs between microhomology stretches (few bps) o Tails are trimmed and gaps are filled through DNA synthesis
- 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
- 46. What is the role of RecBCD in E. coli? It plays a role in homology directed repair, where it has nuclease and helicase activity. It attaches to double stranded breaks and moves along the DNA to unwinding it and degrading it. When it reaches a chi site then nuclease activity decrease and the 5´strand is degraded
- 47. What are Chi sites? Chi (Cross over Hotspot Instigator) sequence is reconcnised by a RecBCD. When RecBCD reaches the Chi site, nuclease activity decreases and degrades the 5´strand.
- 48. Genes involved in repair and recombination are often implicated in some diseases as well, name two example for such a disease. o Werner Syndrome o Bloom Syndrome o Rothmund Thomson Syndrome
- 49. What is the role of RecA (RAd51) in homology directed repair, where else does it occur? It loads onto the single stranded DNA and stimulates single-strand-duplex pairing. Also plays a role in homologous recombination.
- 50. What is the consequence of using homologous chromosomes as templates for homology-directed repair? When sister chromatids are not available (as in G1), homologous chromosomes can be the template for homology-directed repair (These are not identical!) • When a break is repaired from a homologous chromosome, the repair now has the sequence of the homolog, not the original sequence, gene conversion might occur. DNA is changed and is afterwards a copy of the homologous chromosome. This can lead to a loss of heterozygosity, which lead to a loss of one of the two allels.
- 51. Explain how yeast switches its mating type and why this is interesting for Molecular biology? The switching uses homology directed repair. There are three loci the active MAT locus and two silent loci one carrying alpha and one a information. DNA is degraded on both sides by exonuclease. It degrades the DNA for MAT allel. Gap need to be filled. The endonuclease HO cuts in the MAT locus and either the information of alpha or a is copied there potentially changing the mating type. • Several disease-causing microorganisms use gene conversion to change their cellsurface antigens and evade the host immune response
- 52. Does the cleavage of a holiday junction always lead to large areas of recombinant product? No this depends on how the junction is cleaved. · Vertical: exchange · Horizontal: no-exchange
- 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.
- 54. Depict the first steps in homologous recombination · Double stranded breaks, resection to create single stranded DNA · Invasion of a heterodublex by one broken end to form a hetrodublex · DNA is synthesized from the invading strand. · The displaced strand of the D-loop is captured by the remaining 3' end. · DNA synthesis occurs and holiday junctions form 3/41.
- 55. What is the relationship between homology directed repair and homologous recombination? They are similar and the initial processes are almost the same.
- 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
- 57. Describe Break Induced replication · Resection to expose single stranded DNA , 3´overhangs · Invasion of another dublex of one of the three pverhangs, generating heterodublex and a displacement loop, DNA is synthesized · Failure to capture the other end od DNA · Fork reassembles and lagging strand is synthesized · Cleavage · Ligation
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- 58. What are some consequences of Break Induced replication? Potentially loss of heterozygousity as it is only from one strand Potentially this Replication is not as precise as normal replication
- 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