# Transcriptional PBR cycles at pericentromeric repeats cause gross chromosomal rearrangements through Rad52-dependent ADR-loop formation

## TL;DR

This study shows how loss of heterochromatin leads to chromosomal rearrangements through transcriptional cycles and Rad52-dependent DNA repair processes.

## Contribution

The paper identifies a novel mechanism involving PBR cycles and ADR-loops in causing chromosomal rearrangements when heterochromatin is lost.

## Key findings

- Loss of Clr4 increases R-loops at pericentromeric repeats, leading to GCRs.
- Rad52 facilitates ADR-loop formation and GCRs in the absence of heterochromatin.
- RNaseH1 overexpression reduces R-loops and GCRs, confirming their causal role.

## Abstract

Heterochromatin marked by histone H3 lysine 9 (H3K9) methylation represses transcription of pericentromeric repeats, thereby suppressing gross chromosomal rearrangements (GCRs). However, it remains unclear how transcription causes GCRs when heterochromatin is lost. Using fission yeast, we show that transcriptional Pausing–Backtracking–Restart (PBR) cycles accumulate R-loops, leading to GCRs. DNA–RNA immunoprecipitation (DRIP) revealed that loss of Clr4, the H3K9 methyltransferase, increased R-loops at pericentromeric repeats. Overexpression of RNaseH1 in clr4∆ cells reduced both R-loops and GCRs, demonstrating that R-loops cause GCRs. Tfs1/TFIIS and Ubp3, required for transcriptional restart, and Seb1, involved in pausing at pericentromeres, were required for R-loop accumulation and GCRs, implicating PBR cycles in the formation of genotoxic R-loops. We also demonstrate that Rad52 recombinase localizes to pericentromeric repeats and facilitates GCRs in clr4∆ cells. rad52–R45K, which impairs single-strand annealing (SSA), reduced GCRs. A single-stranded DNA (ssDNA) region within an R-loop may anneal to homologous ssDNA to form Annealing-induced DNA–RNA-loops (ADR-loops). Indeed, Rad52 facilitated ADR-loop formation in vitro. Polδ was also involved in GCRs. These data suggest that, when heterochromatin is lost, transcriptional PBR cycles accumulate R-loops at pericentromeric repeats, and Rad52-dependent SSA converts R-loops into ADR-loops followed by Polδ-dependent break-induced replication (BIR), resulting in homology-mediated GCRs.

Graphical Abstract