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  • Our finding that the ATRX

    2020-09-21

    Our finding that the ATRX-DAXX-H3.3 complex promotes a form of HR is likely to have important implications for our understanding of the ALT recombination process that takes place at telomeres. We were surprised to observe that ATRX promotes a form of HR, since loss of the ATRX-DAXX-H3.3 complex is necessary for generating the ALT phenotype (Lovejoy et al., 2012). Thus, it is tempting to speculate that the HR sub-pathway promoted by ATRX-DAXX-H3.3 is detrimental if employed for the maintenance of telomere length. However, telomeric SCEs and other forms of crossovers do frequently occur in ALT-positive, ATRX-negative cells. Moreover, DSBs induced inside telomeric sequences were shown to be repaired by break induced replication (BIR) involving extensive DNA repair synthesis independent of ATRX (Dilley et al., 2016). Thus, either the regulation of recombination at telomeres might be fundamentally different to the recombination processes that take place at non-telomeric DSBs or ATRX has additional roles at telomeres that make a pathway comparison difficult. One such function could be that the loss of ATRX leads to an increase in secondary structures at telomeric repeats, including telomeric repeat-containing RNA/DNA hybrids, which might represent initiating events for the ALT process (Dilley and Greenberg, 2015, Maciejowski and de Lange, 2017). Finally, the ATRX-DAXX-H3.3 complex likely functions during the repair of two-ended DSBs by overcoming torsional stress arising during extended DNA repair synthesis (see discussion below). Since the topological constraints are very different at freely rotatable telomeres, this might also explain differences in DSB repair pathway usage at telomeric versus non-telomeric DSBs. ATRX has known CHIR-124 receptor remodeling functions, for which we uncover a novel and unexpected role during HR. Our findings demonstrate a dependency of DNA repair synthesis progression on the concomitant incorporation of H3.3 by ATRX-DAXX, which is a prerequisite for repair completion by a crossover forming pathway. This tight coupling of chromatin reconstitution with DNA repair synthesis is further supported by the interaction between ATRX and PCNA, which is necessary for the completion of the process. This is analogous to what occurs during replication, where CAF-1 associates with PCNA to direct the deposition of the histone H3.1 behind the replication fork (Shibahara and Stillman, 1999, Tagami et al., 2004). This coordination of chromatin assembly and DNA synthesis is important for the regulation of elongation rate and fork progression (Mejlvang et al., 2014). A function for histone H3.3 has been described during transcription, where H3.3 deposition by HIRA is important for chromatin integrity, and H3.3 exchange promotes transcription initiation and elongation and is linked to transcription rate (Ray-Gallet et al., 2011, Sarai et al., 2013). While it is not fully elucidated, histone exchange appears to be an essential element of DNA/RNA synthesis that regulates, on various levels, replication, transcription, and repair. In our model, H3.3 deposition is required for DNA repair synthesis, which could be important to maintain chromatin integrity over long repair patches and also allow for the movement of the D-loop by providing structural stability. The unwinding of the DNA, as in transcription, generates torsional stress, manifesting as positive and negative supercoiling ahead of and behind the moving synthesis complex, respectively. Positive stress can destabilize nucleosomes and cause histone eviction, while negative supercoiling promotes nucleosome assembly (Gupta et al., 2009, Teves and Henikoff, 2014). Taken together, we propose a model in which histone deposition is tightly coupled to DNA repair synthesis during HR, in which a high histone turnover could manifest as a balance between histone disassembly in front of the moving D-loop coupled to reassembly behind it. The coordination of these processes ensures the timely reconstitution of chromatin to provide topological stability that allows DNA synthesis progression and genomic protection (see Figure S7 for further discussion).