Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Abstract

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

Mechanism of homology search expansion during recombinational DNA break repair

“…To distinguish between these possibilities, we depleted AID-tagged Pol3 alone or in combination with the PCNA clamp loader subunit Rfc1 after S-phase completion ( Figure S8D ). We previously showed in these strains that this single and double depletion strategy led to a 3– and 10-fold decrease of D-loop extension, respectively 9 . Both depletion conditions yielded highly similar results.…”

“…Homology identification leads to NPF collapse specifically at the homology-containing end. This collapse is likely to limit coincidental homology identification at multiple genomic sites along a single Rad51-ssDNA NPF, and thus precludes the cascade of repeat-mediated chromosomal rearrangements initiated by multi-invasion DNA joint molecules 6,9 .…”

“…Inhibition of POL3-iAID expression and depletion of Pol3-iAID independently or together with Rfc1-AID-9Myc was conducted similarly to 9,96 . Transcriptional inhibition was induced upon 50 μg/mL doxycycline and protein depletion induced upon 1.5 mM IAA addition 1 hour after S-phase release and HO induction, to allow for bulk DNA replication to take place.…”

“…Cell fixation is adapted from Micro-C-XL 100 . Approximately 1.5×10 9 haploid cells were fixed with 3% formaldehyde (Sigma-Aldrich, cat. F8775) for 30 minutes at RT at 120 rpm.…”

“…The molecular mechanism of dsDNA sampling, mainly defined for bacterial RecA-ssDNA NPFs in vitro 2 , occurs at the level of a few RecA monomers and is multiplexed along the length of a single NPF 3,4 . The conservation of this inter-segmental search mode in eukaryote is supported by the ability for a single NPF to form D-loops and multi-invasion DNA joint molecules using internal regions of homology in S. cerevisiae cells, and with reconstituted yeast and human NPF in vitro 5–9 . Accordingly, internal homologies are efficiently used as repair templates in S. cerevisiae 10 .…”