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THE DAMAGE CHECKPOINT, is there a way in whcih we can prevent tumor…

- - - - HU
      - TYMIDINE
    - - agents inducing DNA damage
        
        DNA DAMAGE BLOCKS DNA SYSNTHESIS
        
        RADIATIONS
        
        X-rays caused chromosome breaks (DSBs in DNA) and cell cycle blocks in G2
        
        by exposing cells to radiations you observe a delay in the cell cycle transitions
        
        AT patients
        
        hypersensitivity to radiations
        
        they observed a high accumulation of chromosome alterations, especially if exposed to X-rays
        
        normal cells: the incorporation of DNA synthesis precursors decreases
        in AT cells: continue the DNA synthesis process
        
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        RDS
        
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        Epistatic groups
        
        people started to study different classes of mutants in model organisms causing sensitivity to radiations
        
        these epistatic groups were reflecting the activity in different types of DNA repair pathways
        
        1st gene cloned → rad52
        
        very important for DSB repair and cells without rad52 accumulate a lot of chromosomes breaks
        
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        we block the cell cycle in a specific phase and then we put MMS that will cause a damage and block DNA synthesis
- - - - it controls global events ➜ it is eventually silenced when the local damage is removed and recovered and the cell can start normal metabolism
        
        in case of a huge number of damage, or damage that cannot be repaired cells accumulate damage and activates some extremes responses
        
        APOPTOSIS
        
        Best way for the cell to control cell proliferation in case of irreparable damage.
        
        PROGRAMMED CELL DEATH.
        ▸ IT IS A GENETIC PATHWAY THAT LEADS TO DAMAGED CELLS TO BE REMOVED FROM THE TISSUE
        
        induced by a signal and can be programmed during the development (allowing the correct formation of the tissue/organ)
        
        ➔ if apoptosis doesn't work very well in a damaged tissue the cell may become cancer or immortal cell allowing tumorigenesis
        
        the apoptotic pathway is controlled by protein kinase B (AKT)
        ⇛ many stimuli in the environment and metabolism
        ⇛ PIKK turned in to PIP3 that activates it
        ⇛inhibits the apoptotic pathway
        
        STRONG EARLY INHIBITOR OF THE APOPTOTIC PATHWAY
        
        the ATK pathway controls the Tor pathway
        
        P53
        
        critical factor responding to damage activating apoptosis in higher euk cells
        
        it controls gene expression, so it controls the transcription of factors involved in apoptosis
        
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        G1 / S TRANSITION
        
        in case of damage
        
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        S / G2 TRANSITION
        
        in case of damage
        
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        SENESCENCE
        
        cells stop proliferating going though senescence
  - - - HO is used to induce the DSB in a yeast cells where only a chromosome was present in 2 copies.
        
        They introduces another chromosome containing a specific gene (ADE3), these cells would accumulate red pigment and appear red.
        
        In presence of Gal the HO expression is induced, it causes the DSB and the DDC is activated .
        The cell remains blocked thanks to the rad9 pathway.
        
        thanks to the rad9, that is important to activate the DDC in response to damage the cell stays blocked for a while but then it restarts the proliferation EVEN IF THE DAMAGE COULD NOT BE REPAIRED
        
        They prevented the repair of the chromosome by the introduction of KO of RAD52, involved in HR.
        that is why the repair was irreparable and stayed there forever
        
        However the cell would restart to produce a colony after a while of WHITE cells
        
        this is becasue they have lost the extra chromosome and by not having the ADE3 anymore the cells appear to be white
        
        If we look closely though we see that they are RED IN THE CENTER
        
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- - - - to understand how these two networks interact and how they influence each other
        
        DSB-BASED SYSTEM
        
        system that can induce DSB at a very precise site
        
        we can follow the locally occurring events (break formation repair and restoration of cell division)
        
        the globally occurring events (activation of the DDC and recruitment of its factors)
  - - - NHEJ
        
        the ends are shortly modified or simply re-ligated to join them
      - HDR
        
        the ends are extensively processed, it requires a homologous sequence (donor) to repair the break
      - BER
      - NER
        
        one fundamental repair pathways
        
        ❖ specific factors detect the DNA base alteration
        ❖ a nuclease cuts the DNA around the lesion
        ❖ the DNA containing the lesion is removed by a helicase
        ❖ DNA pol will fill the gap
        ❖ the gap will then be closed by ligase
        
        Fundamental proteins involved in repair mechanisms
        
        helicases: enzymes that can just process single strand DNA.
        They can move in one direction or the other using ATP
        
        exonuclease: protein that degrade the DNA both from the 3' and 5' ends, they have different polarity.
        They cannot move in both direction, just one and are ATP dependent enzymes
      - MMR
      - FANCONI ANEMIA PATHWAY
        
        The Fanconi Anemia
        
        associated with the accumulation of β strand and mutations in genes involved in this DNA alteration repair
        
        alcohol metabolism
        
        critical metabolism that causes the accumulation of these β strand and intra-β crosslink
        
        acetaldehyde is accumulated and it is very toxic to neurons and cause the accumulation of DNA alterations
        
        can be solved by the Fanconi anemia pathway
        
        very complex pathway that involves many FANC genes
        
        each gene plays a different role in the pathway and have been found to code for known proteins involved in mechanisms of DNA repair
        
        if these genes are mutated:
        
        genetic instability
        
        cancer predisposition
        
        sensitivity to agents that cause the inter-strand cross-linked DNA structure (ICL)
        
        ICL
        
        molecules will tend to form covalent bonds from one filament to the other in the double helix
        
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        ICL repair mechanism
        
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  - - - when oncogenes are activated sometimes cells respond as a consequence of this activation by inducing a lot of transcription, and we can have the amplification in terms of expression of many genes
        
        transcription-replication conflicts → stressful response and probability of different types of damage to occur
  - - - type of analysis used to identify the group of genes controlling a particular process or pathway
      - we select two alleles (a and b) that are sensitive to ionizing radiations
        
        we can study the behaviour by comparing the wt with a, b and the double mutant , and we can see if they affect or are cooperating in the same pathways of repair
    - - they will directly cut the double strand
        
        the cells → filter mask and ionizing radiations pass though the holes damaging only specific sites in the nucleus, while the other parts are protected
        
        visualize the recruitment of specific factors at the lesion level in vivo
    - - it is a system that tries to replicate the DSB based system used in yeast with HO but using I-sce1 ➜ a specific RE that is able to cut a specific site in human cells
        
        another RE that can be used is AsiS1.
        is able to cut many sites in the genome but just 18/19 are known.
        
        with this tool we can then visualize the kinetics of the mechanism
    - - we can target a specific sequence providing a specific and precise sgRNA
        
        we can induce a DSB in that point and study what happens
  - - - many of these factors involved in the checkpoint and in the repairing are recruited at the damaged site
        
        ➤ at the level of the damaged chromatin transcription is normally silenced since the basic TF machinery is excluded
      - they reconstituted the kinetics of all the factor recruitments at the foci level in human cells
        
        damage induced in the big nucleus of the cell with UV light inducing DSB
        
        after the formation of the strip, they saw that its shape would change quite a lot becoming thicker and then it disappears after the chromatin restoration
        
        ATR is present at the center of the strip
        
        ATM is much more present at the periphery
        
        laser has a lot of energy and it would create alterations in the flanking regions
        
        THE ENDS STATE
        
        we can have CLEAN ENDS just in case of DSB induces by endonucleases or Cas9
        In this case they would not be modified and we would have the break at the P-diester bond on both ends
        
        we can also have BLOCKED ENDS that are blocked by proteins
        → many proteins are recruited at the clean ends so they block them preventing the access of other proteins that could be involved in processing
        
        topoisomerases
        
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        DIRTY ENDS
        can be formed by ionizing radiations or other chemical reactions
        → the sugar and the P backbone are completely altered
        → very dangerous since the enzymes that process the clean ends cannot process them because they do not recognize them
        
        they should be transformed into clean ends and accessible by the enzymes at the process the ends
        
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        dirty ends
  - - - events that occur locally at the level of DSB depends on how the chromatin is remodelled
        
        It can be wrapped around the histone core forming the nucleosome
        
        regulations though post translational modifications of histones
        + histone variants
        
        it can be in the unfolded tails
        
        subjected to different enzymes that modify specific residues
        
        the nucleosomes are subjected to different types of movement
        
        specific enzymes can move the histone core
        
        slide them
        
        completely remove them
        
        substitute them with variants
        
        not spontaneous reactions
        
        can completely reshape the chromatin around the lesion allowing the recruitment of DDR factors
        
        Nucleosome Memory
        
        histones can be modified, recycled and reused after the repair
        
        Steps of the process
        
        1.access/prime stage
        
        PARYLATION
        
        ➜ PARP1 and PARP2
        
        PARP1 is recruited at the DNA lesion and modifies the histones
        
        concomitant with the recruitment of PIKK and ATM kinase that phosphorylates the flanking nucleosomes to the DSB
        
        some nucleosomes can be removed
        ➜ free space for the enzymes involved in repairing
        
        in depth: PARYLATION
        
        role in the DNA damage
        
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        PARP1
        
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        2. repair and checkpoint activation
        
        the cell cycle is blocked
        
        the damage is repaired
        
        everything is restored back
        
        3. recovery stage
        
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    - - in response to the formation of DSB all the chromatin in the nucleus is decompacted (→ nucleosome sliding, histone cores are disloged or degraded)
      - The nucleus interacts with the actin filaments in the cytoplasm
        
        actin filaments interact with specific nuclear pore structures
        
        allows the nucleus to be shaken
        
        this allows all the chromosomes to be mixed in the nucleus facilitating the movement of the broken chromosomes and the pairing of the homologous sequence with HR
        
        ➜ THE DNA DAMAGE RESPONSE IS ACCOMPANIED BY A DEEP CHANGE IN THE NUCLEAR ARCHITECTURE AND PHYSIOLOGY
        
        another example of this
        
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- - - - mutation of factors involved in the G2/M transitions
        
        Wee1 and ATR kinases