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HDR - Coggle Diagram

- - - - depends on:
        → nucleases and helicases
        → factors that allow the annealing such as rad52 or recombinases like rad51
    - - when the 3' tail is formed and prepared for recombination, with the invasion of the homologous sequence it can happen that they are not perfectly complementary
        
        the replication is not possible since the polymerase cannot bind
        
        cells have to remove this non homologous tail
  - - - depends on:
        → ssDNA binding factors, recombinases, strand exchange and annealing factors as well as regulatory factors
  - - - depends on:
        → nucleases and helicases
        → topoisomerases and ligases
- - - - RPA COMPLEX
        
        small complex formed by replication factors 1, 2 and 3
        
        it covers the ssDNA protecting it
        
        creates a platform for the recruitment of other proteins
    - - this favours HDR, SSA and MMEJ
        
        big impact on genome stability, in fact this fist steps are finely regulated because it can lead to rearrangement of the chromosomes
    - - 1
        
        in bacteria
        
        RecBCD pathway:
        exposition of the key sequence allowing the HR, so the strand invades the sequence
        
        RecQJ pathway :
        the helicase (RecQ)opens the ends of the DSB separating the filaments. RecJ attacks the exposed 5' filament and removes all the bases leading to resection, so the other strnad is now a single strand that is ready for recombination
        
        in humans
        
        a exonuclease digests one filament removing the bases and exposing the ss 3' strand
        
        a helicase separates the two filaments exposing the 5' termination tail and a nuclease processes this tail by cutting the filament in pieces so exposing the ss 3' end
      - 3 nucleases are involved
        
        1. one is dependent of the MRE11 complex
        2. Exo1
        3. Dna2
        
        MRE11 complex
        
        is a 3' to 5' exonuclease and a site-specific endo-nuclease
        
        3 subunits
        
        nuclease subunit → Mre11 itself with the phospho-esterase domain
        
        interacts with rad50 that possesses an elongated structure called coiled-coil formed by α helicases
        
        1 more item...
        
        Nbs1 (in human) / Xrs2 (in yeast)
        
        it is present at the head of the complex and it contains the fork head domain (PHA)
        
        interacts with Mre11 and PIKK complex (ATM)
        
        interacts also with CtlP,
        important because it possesses a nuclease activity
        
        basic activities
        
        ENDONUCLEASE ACTIVITY
        
        ➜ Localized at the level of the DSB ends
        ➜ the CtlP is activated and cuts one filament which is 5' ended
        ➜ this is the NICKASE ACTIVITY
        
        ➜ then it starts RESECTION
        ➜ as an exonuclease it moves 3' → 5' direction reaching the ends of the break
        
        STRUCTURAL ACTIVITY
        
        thanks to its coiled-coil structure it helps maintaining the two DSB ends together on both sides (similar function of KU in the NHEJ)
        
        NICK MODEL
        
        After the break is formed the MRE11 together with the CrlP is recruited 100bp from the break
        
        the complex is activated by the CtlP that nicks the strand on both sides of the break
        
        At the nick level the MRE11 starts resecting towards the DSB end ➜ SHORT - RANGE RESECTION
        
        Exo1 and Dna2 together with ReQ helicase move in the opposite direction ➜ LONG - RANGE RESECTION
        
        after this mechanism a 3'ending single strand is created and immediately covered by RPA
        
        gives a way to process blocked ends
        
        SENATAXIN → critical role in cleaning the DSB ends
        
        dismantles the R-loop structure so MRE11 can start processing the strands
        
        can MRE11 process the strands and make the nick anyway? yes, it sis just more efficient because otherwise we can have the accumulation of KU that will trigger NHEJ and we can have translocations
        
        REGULATION OF THE RESECTOSOME
        
        The resectosome is a very dynamic complex that has a lot of associated factors.
        all these factors can be post-translationally modified by different enzymes and they can so influence its activity
        
        CDK
        
        1 more item...
        
        Exo1
        
        2 more items...
        
        Dna2
        
        1 more item...
        
        Mre11
        
        1 more item...
        
        in the cell cycle
        
        because of the fact that the MRE11 complex is able to nick the DNA at the level of the DSB thanks to the activity of the CDK that activates the CtlP that nicks the DNA ➜ the process is active mainly in S/G2 phase because the CDK1 is active there
        
        1 more item...
        
        PIKK (ATR and ATM)
        
        DDC is activated by the resectosome
        
        BUT the DDC also limits DSB resection
        
        1 more item...
        
        Mec1 activation
        
        is a kinase whose activity is stimulated at the damage site by the interaction by Ddc2, Dbp11 and 911
        
        3 more items...
      - how can DSB resection be monitored (in vivo) ?
        
        Indirectly:
        we can test the binding of RPA to a DSB by Chip
        or the co-localization of RPA to gamma-H2AX
        
        Directly:
        with PCR protocols
        after the break if the chromosome remains double stranded we can amplify the sequence using specific primers. ➜ Even if the strand is a single strand and primers are applied we and in both cases amplify the sequence
        HOWEVER, before doing the reaction the target sequence is digested with RE (CAN CUT JUST DS) and if it cuts between two primers we have no product
        So if we have a single strand, RE cannot cut it so the sequence is amplified
        ➜ we will then know if we have resected DNA because our DNA sequence will be amplified, otherwise it is still double stranded
        
        Directly:
        DNA-combing
        the DNA is extracted and plated on a glass slide and are then with a comb they are distributed and aligned.
        In this was with some tricks it is possible to visualize ssDNA strands under the microscope, so if they are resected we can visualize them and measure their lenght