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NHEJ - Coggle Diagram

- - - - since KU is not present, the two ends cannot be tied together so they start moving and are exposed to the processes that might end up expose micro-homologies ➜ alt-NHEJ MMEJ
        This can lead to cis joining (deletions)or trans joining (translocations)
    - - no translocation → normally even if inaccurate the joining occurs in cis
        rarely can cause translocations since it happens in trans
      - if we have two breaks in 2 different chromosomes
        KU is normally deposited at the end of the break and forms a sort of bridge between the two parts allowing for NHEJ events to occur
        
        KU is an oncosuppressor gene
        
        it maintains the synapsis between the two ends of the DSB suppressing or limiting the events that lead to movement
        
        suppressing translocations
        
        protecting them from deletions
  - - - this causes the fusion of 2 genes : BRC + ABL that lead to leukaemia
        
        the translocation is induced by alt-NHEJ and at the break point there is the annealing between part of the chr22 and chr9.
        The two parts then re anneal because of complementarity and the two ends are reapired though MMEJ
  - - - mutations in this gene cause 2 rare but severe neurological disorders associated with different types of ataxia → AOA2 and ALS4
      - this helicase is able to separate the filaments of the DNA double helix (as typical) but especially it separates filaments between a DNA and a RNA filaments
        
        ➜ involved in the termination of transcription
        (→ but also other activities concerning the DNA and the RNA)
        
        involved in DSB repair
        ⇒ DNA-RNA molecules could be formed during certain repair mechanisms
        
        after the DSB is formed , the cell synthesize a short RNA close to the ends → R-loop formation and sen1 normally removes these transcript leaving clean ends that can be processed either by NHEJ or by HDR
        
        without this helicase the transcript remain there and this may lead to translocations with alt-NHEJ or deletions with in cis c-NHEJ repair
  - - - 1. Strand pairing: a double strand is formed by the annealing of two complementary strands
        2. Pairing + Exchange: we have a circular strand and a double strand with end (one of the strands anneals to the complementary circular one)
        3. Strand Invasion: a strand anneals to the target homologous sequence of the double helix
        4. Strand Exchange: between double strand DNA ends (a strand of the double helix is exchanged with a strand of the double circular DNA)
        
        all these mechanisms are not spontaneous and are instead stimulated by specific proteins
    - - a small protein that forms a complex formed by 11 subunits
      - ring-like structure → the DNA wraps around it
        
        it interacts with ssDNA and allows the annealing and pairing of two strands coming from 2 chrs or from the 2 sides of the break
      - steps
        
        rad52 mediates the annealing of the 2 DNA strand that is case of homology will anneal
        
        the nuclease will cut the flaps (the ssDNA that are not homologous)
        
        the short gasp are sealed with polymerases and ligases
        
        → this system allows the restoration of the intact chromosome but at the end it will be shorter since some sequence will be deleted
        
        RESULTS IN SMALL DELETIONS → NOT A CONSERVATIVE REPAIR
    - - → HO induction, so break formation
        → Southern blot with probe to see the break
        → Over time we can see the appearance of the bad (break) and how it changes during the repair mechanism
        → the cut band disappears and the the product appears after a while
        → the sequence changes also after the repair
  - - - allows the complete replication of an entire arm of the chromosome until the telomere
        
        this happens though the formation of a replication fork
        
        outcome
        
        the donor chromosome remains untouched while the broken one is completely new.
        we will have a part of the chromosome is still the same while the other is completely reconstituted as a result of the copy of the replication
        
        THE REPAIRED CHROMOSOME IS NOW EQUAL IN PART TO THE DONOR CHROMOSOME
        ➜ THERE IS LOH (loss of heterozygosity), that is one of the robust characteristics of the diploid genome
        
        leads to half-CO or no-CO
    - - the invasion and the pairing is rejected after a while and the newly synthesised sequence re-anneal with the broken chromosome
        
        no-CO
    - - after the invasion on the donor template the synthesis goes on but then the other end on the other side is also captured (second end capture)
        
        two types of joining molecules on the 2 sides → dHj
        
        CO or no-CO
- - - - typical of Burkitt lymphoma (BI)
      - one part of the chromosome translocates to the other chromosome leading to a reciprocal translocation
      - this translocation happens also with other chromosomes, not just with chr 8
        ➜ the particular locus encoding for IgH is a hotspot for recombination since this locus is exposed to programmed DSB formation
        this locus is the VDJ locus
        
        NHEJ is involved in the repair of the DSB created during the VDJ recombination and CSR in lymphocytes maturation
        if we have mutations in the NHEJ factors → prevent Ig genes maturation leading to severe immunodeficiency
        
        Chr 14: IgH locus: cassettes formed by different repeats
        → V segments
        → D segments
        → J segments
        → constant region exon
        
        V segments are rearranged in a way in which just one cassette is joined to another cassette + the variable regions exons.
        The gene is then transcribed and the typical Y structure the antibodies is formed
        
        the tip part is variable → many types of antibodies
        
        high variability ➜ reached by the rearrangement of these segments in different ways but happens in very specific steps
        ➜ D is joined to J and then this part is joined to the V part
        
        VDJ RECOMBINATION
        
        RAG nucleases
        
        1 more item...
        
        after the VDJ if formed it is then joined to the constant region
        the presence of one C part or another determines if
        
        the antibody is localized in the membrane
        
        is is secreted in the body
        
        it is secreted outside on the surface
        
        Now we know that this process involves DNA-PK and the KU complex as well as the LIG4
- - - - Sometimes the joining of the 2 parts could lead to some imprecise joining because if there is no homology between the two parts a few bases have to be removed or added to the break point
        
        NHEJ allows to reseal a break but it is not always very accurate
        
        error - prone
    - - 1) KU heterodimer (acts as a docking station for loading other NHEJ factors)
        2) DNA-PK
        3) Polymerases
        4) XRCC4, XLF and LIG4
        5) PAXX
    - - the imprecise case, the more frequent, exposes the cell to accumulating variations at the break point
        
        worse situation ➜ when there are 2 DSB in 2 different chromosomes
        
        there could be erroneous repair,
        
        and in this case 2 different loci are altered
        
        one arm of the chromosome can be resealed with another arm of the chromosome → formation of an acentric chr and a diacentric chr ( cells die usually)
        
        reciprocal translocation (as in Bl)
        
        formation of one-ended DSB during replication
  - - - joining can be mediate by microhomologies
        
        Micro-Homology Mediated End Joining (MME)
        
        allows the re-join of the ends thanks to the exposition of a short region of homology on both sides of the break
        
        leads to severe rearrangements since now we will have extensive deletions
        
        the entire part after the resection of the ends, and after the action of the ligase, is lost
        
        completely KU independent
        
        BUT requires PARP polymerase that will create the scaffold structure for the recruitment of the nucleases (MRE11 complex)
        
        LIG4 is also not recruited
        
        they recruit LIG1 and LIG3
      - the MRE 11 complex is recruited at the ends from PARP1 and performs the resection