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  • ATRX belongs to the switch sucrose non fermenting

    2020-06-30

    ATRX belongs to the switch/sucrose non-fermenting (SWI/SNF) chromatin remodeling family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression (Gibbons et al., 1995). Most of these mutations are located in two highly conserved regions of the ATRX protein: a C-terminal ATP-dependent motor domain and an N-terminal chromatin-binding ATRX-DNMT3-DNMT3L (ADD) domain that includes a plant homeodomain (PHD) zinc finger (Gibbons et al., 2008). The alternative lengthening of telomeres (ALT) pathway is a HR-associated process that is activated in 10%–15% of all cancer alisertib to maintain telomere length independently of telomerase activity (Dilley and Greenberg, 2015). Mutations in the ATRX-DAXX-H3.3 complex have been found in a variety of cell lines and tumors with the ALT phenotype, leading to the suggestion that this complex functions to suppress the ALT pathway (Lovejoy et al., 2012, Maciejowski and de Lange, 2017). ATRX also functions during replication, as ATRX knockdown cells show hypersensitivity to agents inducing replication stress, exhibit perturbed S-phase progression, and fail to properly handle stalled replication forks (Leung et al., 2013). Collectively, this suggests that ATRX regulates recombination processes although it is currently unclear how this is achieved.
    Results
    Discussion HR of two-ended DSBs can proceed by two different sub-pathways. One of the pathways involves extended DNA repair synthesis, the formation of a dHJ, and crossover formation, while the other HR sub-pathway, SDSA, prevents the formation of a dHJ by displacing the invading strand from the donor sequence. Here, we provide evidence that the ATRX-DAXX-H3.3 chromatin remodeling complex promotes the dHJ mechanism at the expense of SDSA (see Figure 7). In our study, we have specifically analyzed G2-phase cells to avoid potential complications from replication-associated breakage processes. Thus, we investigated the repair of two-ended DSBs, which can be repaired by either SDSA or a dHJ mechanism, and did not consider repair pathways that deal with one-ended DSBs. The observed diminution of SCE formation after ATRX depletion and the partial dependency of the ATRX-DAXX-H3.3 pathway on Mus81 argue that crossovers can arise during repair by this pathway. Since a positive correlation between the length of DNA repair synthesis and crossover formation was previously described (Cole et al., 2014, Guo et al., 2017), the ability of ATRX to promote the appearance of detectable BrdU foci and the fraction of LTGC events in a reporter system supports the model that ATRX-DAXX-H3.3-dependent repair represents a dHJ mechanism. HR in the absence of the ATRX-DAXX-H3.3 complex takes place in different cells to varying degrees. In our assays, ALT-positive U2OS cells showed the highest level of Rad51-dependent, ATRX-independent HR, but ALT-positive Saos-2 cells and ALT-negative HeLa cells were capable of performing this process, which we argue represents SDSA. This speculation is supported by the observation that HR alisertib without ATRX does not give rise to SCEs and does not require Mus81. Moreover, premature termination of DNA repair synthesis due to ATRX-DAXX-H3.3 deficiency is most compatible with an SDSA pathway that often encompasses gene conversion tracts of less than 50 bp, while crossover-forming pathways exhibit gene conversion tracts on the order of 500 bp (Elliott et al., 1998, Cole et al., 2014). These estimates are consistent with the findings that the frequency of LTGC events with a tract length of >1 kbp increases in the presence of ATRX (Figure 2E), while the frequency of SDSA events with a maximum tract length of 100 bp decreases in the presence of ATRX (Figure 2F). Moreover, the formation of BrdU foci, which are estimate to represent repair synthesis patches of at least 1–2 kbp (Figure S3E), is strictly dependent on ATRX. Since many cellular studies on HR pathway usage have been performed with ALT-positive, ATRX-deficient cells (often U2OS cells), this explains the widely held belief that SDSA represents the major pathway for repairing two-ended DSBs (Heyer, 2015, Haber, 2016). Our study, in contrast, shows that an appreciable fraction of two-ended DSB repair proceeds by a crossover forming pathway if the ATRX-DAXX-H3.3 complex is present.