ATRX and RECQ5 define distinct homologous recombination subpathways

Abstract

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

The Multiple Facets of ATRX Protein

“…The latter pathway does not entail the formation of dHJ and crossover products. These evidence are contradictory with the well accepted role of ATRX in the suppression of HR in telomeric regions suggesting that the regulation of recombination sub-pathways might be fundamentally different in genomic and telomeric regions [ 44 , 123 ].…”

“…In order to stimulate HR-mediated replication fork recovery and DSB repair, the ATRX/DAXX-MRN-FANCD2 supercomplex assembles on chromatin and promotes the deposition of histone variants H3.1 and H3.3 and recruits HR and replication restart key factors such as RAD51 [ 42 , 122 ] and CtIP [ 41 ]. A recently published paper suggests that ATRX (together with DAXX) takes part in DNA two-ended Double-Strand Break (DSB) repair inhibiting RECQ5 mediated Synthesis-Dependent Strand Annealing (SDSA) and promoting a different HR subpathway [ 44 , 123 ]. This process involves extended DNA repair synthesis, D-loop formation, the creation of dHJ, and their resolution by the nuclease Mus81 potentially generating Sister Chromatid Exchanges (SCEs) [ 124 , 125 ].…”

“…This process involves extended DNA repair synthesis, D-loop formation, the creation of dHJ, and their resolution by the nuclease Mus81 potentially generating Sister Chromatid Exchanges (SCEs) [ 124 , 125 ]. In this model, ATRX performs its function at later stages of HR, after RAD51 removal by RAD54 [ 44 ] competing with RECQ5 and determining the HR subpathway [ 123 ]. DNA repair synthesis progression is closely coupled with incorporation of H3.3 by ATRX/DAXX complex, which is essential to overcome topological constraints at the moving D-loop.…”

Homologous Recombination Subpathways: A Tangle to Resolve

“…We have shown that the chromatin remodeler ATRX promotes this subpathway of HR that involves long stretches of DNA synthesis leading to the formation of a high frequency of COs visualized as sister-chromatid exchanges (SCEs; Juhász et al, 2018 ). Interestingly, the HR intermediates formed by this pathway, which can be detected as IR-induced ultra-fine bridges (UFBs), are completely dependent on the structure-selective nucleases MUS81 and GEN1 for resolution and are independent of BLM function ( Elbakry et al, 2021 ). Since BLM has a well-documented role in suppressing endogenous SCEs, HR at two-ended DSBs can lead to distinct structures than those formed at replication-associated lesions that may not be classic dHJ and are therefore processed differently.…”

“…The regulation of pathway choice seems to be dependent on proliferating cell nuclear antigen (PCNA) interaction, as both ATRX and RECQ5 possess PCNA-interaction peptide (PIP) domains that are essential for their HR function. Repair studies using mutants suggest that ATRX and RECQ5 compete for PCNA binding, possibly involving post-translational modifications (PTMs) that could regulate the downstream processes ( Elbakry et al, 2021 ). The possibility of PTM-mediated regulation would be consistent with a role of RECQ5-dependent PCNA ubiquitination as well as PCNA-SUMO2 conjugation during transcription-replication conflict resolution, which serve to remove PCNA and RNA polymerase II from chromatin, respectively ( Urban et al, 2016 ; Li et al, 2018 ).…”