Fotoluminiscencia de nc-Si embebidos en a-Si

MMBIR is based on the modification of BIR mechanism for one-end double stranded break repair (Figure 1-8C) [15, 101]. The distinctions between them is that BIR is RecA/Rad51-dependent because it includes an invading 3' end into a homologous repair partner. Thus, BIR is known to be limited when RecA/Rad51 is down regulated.

By contrast, the annealing event of the MMBIR model requires only microhomology sequence to complete DNA repair and thus is a RecA/Rad51-independent pathway. identical sequences to repair damaged sequence. If a damaged region is repaired using homologous sequence in the same chromosomal position of the sister chromatid or the homologous chromosome, the repair is error-free without any changes in DNA structure. However, in some occasions, the improper repair might utilize homologous sequences in different genomic/non-allelic positions. The process is termed non-allelic homologous recombination (NAHR) (Figure 1-9) [15]. NAHR is a form of homologous recombination that occurs between two non-allelic homologous sequences. NAHR mostly occurs within low copy number repeats (LCR) where sequences bear >95% identity homologous elements through the human genome.

These events are responsible for a wide range of disorders. Misalignment of LCRs during NAHR is an important mechanism underlying CRs. NAHR is responsible for translocations, deletions, inversions and loss of heterozygosity (LOH). NAHR causes crossing over in a recombination repair event using a direct ectopic repeat on the same

Figure 1-9. Generation of either duplication or deletion rearrangement by NAHR.

Each line represents a single DNA strand. The substrates and products of recombination are shown.

NAHR can utilize two non-allelic homologous sequences (block A and B) as substrates for recombination on the same (A) or sister, homologous chromosome (B). (A) The high homology region are depicted as blue rectangles with different shades of blue, Block A and B misalign in direct orientation (shown by arrows), and subsequently cross over. The end result with the deletion of the sequence between the two blocks is shown as a two-tone blue junction fragment. (B) NAHR can also occur by unequal crossing over if a recombination event uses a direct repeat as homology on sister, homologous chromosome (or occurs between two lengths of DNA that have high homology sequence, but are not allele). A crossover outcome leads to products that are reciprocally duplicated and deleted for the sequence between the repeats (y, red block).

chromosomal template (Figure 1-9A). It can lead to reciprocally duplicated or deleted genes within these repeats (Figure 1-9B). A crossing over between different chromatids carrying the same alleles can result in heterozygosity from extensive translocations. Altogether, when non-allelic homologous recombination occurs between different highly similar sequences during meiosis, this event might give rise to genetic disorders since it causes the loss or increase of the genetic martial. In mitotic (somatic) cells NAHR can cause rearrangements of various types which are common in cancer. Thereby, while homologous recombination is a vital basis of DNA repair mechanisms, it is also regarded as hazardous when unscheduled DNA repair mechanisms are executed by NAHR.

1.3.4 Other cellular responses in the maintenance of genome integrity

Chromosomal structural changes can be induced by an incorrect choice of homologue partner leading to rearrangements. Cells prevent such changes by avoiding the faulty recombination events through several different regulatory pathways. First, at the chromosomal level, cohesins play a vital role for the accurate reparation of DNA.

Cohesin is a protein complex that holds sister or homologous chromatids together and regulates their separation during mitosis or meiosis, respectively [102]. Cohesin is cleaved at the time of anaphase onset (in mitosis) and meiosis II (in meiosis) and is then dissociated from the chromatids. While the cohesins still bind the two chromatids together, they are, nevertheless, assembled at DSBs keeping the two ends of a single DSB together. This facilitates the accurate damage reparation using a sister/homologue chromatid as the preferred partner. Cohesin also provides a physical structural barrier to restrict the opportunity for the utilization of either the intrachromosomal or interchromosomal templates through NAHR, which are susceptible to genetic instability.

Secondly, at the DNA level, there is another reparation pathway to avoid DNA mismatch pairing: DNA mismatch repair (MMR), which can be a barrier to homologous recombination (i.e. recombination between nearly identical sequences) [103-105]. MMR excises the wrongly incorporated or damaged (e.g.

miss-incorporation of bases, erroneous insertion, and deletion) and replaces them with the correct nucleotide during DNA replication and recombination, as well as repairs some forms of DNA damaged bases (e.g. removal of UV induced damaged bases-thymine dimers are repaired by NER). The removal process involves a few or up to thousands of base pairs of the newly-synthesised DNA strand. Such reparation reduces the chance of arrested forks, thus reduces the likelihood of an incorrect restart.

1.3.4.2 Checkpoint activation in response to DNA damage and replication stress In order to maintain the integrity of the genome, cells also utilize surveillance and checkpoint signalling pathways to react to the replication perturbations (e.g. ssDNA or DSBs) [70-72]. The replication checkpoints are the prime defence barriers against replication fork instability. The procession of checkpoint signalling pathways aid the pausing of replication forks temporarily and ensures that the DNA replication resumes/restarts at the normal level. The replication checkpoints are activated through intertwined networks of sensors, mediators and effector to detect, transmit and amplify the damage or replication stress signal. The DNA damage is detected by the checkpoint sensors, following activation of the mediators and phosphorylation of effector kinases. The effector kinases transmit the signals to their downstream target proteins leading to cellular responses (e.g. cell cycle arrest, apoptosis, inhibition of origin firing, stabilisation of replisome associated with DNA and regulation of DNA

repair). There are two key checkpoint sensors to initiate the intra-S phase checkpoint [106]: Ataxia telangiectasia mutated protein (ATM) [74-75], and Ataxia telangiectasia and Rad3-related protein (ATR) [74, 76-77] (Table 3). They are implicated to play a role in different disturbed replication situations. The ATM pathway responds mainly when a replication fork encounters a DSB. Whilst ATR pathway detects stalled RFs where exposed ssDNA regions, the progression of ATR pathway can inhibit fork reversal. For the ATM pathway, the MRN mediator complex, composed of Mre11, Rad50 and Nbs1, are recruited at the DSBs. These recruited proteins can activate ATM and this is thought to arrest the fork before the DSBs, thus preventing further progression of recombination structure generation. In contrast, ATR pathway is activated by the exposed ssDNA regions when the replisome dissociates from the DNA. Once ssDNA–RPA is formed at the collapsed replication forks, two checkpoint mediator proteins requites: ATR-interacting protein (ATRIP) and checkpoint clamp loader Rad17. Rad17 loads the proliferating cell nuclear antigen (PCNA)-like checkpoint clamp Rad9–Rad1–Hus1 (9-1-1complex) [78]. The 9-1-1 complex is then further phosphorylated by ATR and amplifies signals for checkpoint activation.

Following ATM and ATR activation, they then trigger the recruitment and activation of mediator proteins to the site of the DNA damage through the phosphorylation of the effector checkpoint kinases Chk2 and Chk1. Finally, the activation of the effector kinases promotes the checkpoint response through the phosphorylation of targets protein for different, specific processes (including cell cycle arrest, DNA repair procession, and etc.). When checkpoint pathways do not act properly, aberrant structures, such as collapsed and regressed forks will be accumulated. These intermediates are likely substrates for the chromosomal rearrangements by the inappropriate or unscheduled DNA reparation.

An overview of checkpoint signalling. During replication stress, damage is recognized by the checkpoint sensors and through activation of mediators transmit the signal to effector kinases by phosphorylation events, eventually generate full checkpoint response. The main factors involved in the DNA damage and replication checkpoints in humans, S. pombe and S. cerevisiae are shown.