DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres

Abstract

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2-Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

“…Previously, we have shown in fission yeast that loss of Rad51 reduces gene conversion between centromere inverted repeats and increases isochromosome formation in a manner dependent on Mus81 crossover-specific endonuclease (Nakamura et al, 2008;Onaka et al, 2016). The rad52-R45K mutation in the Nterminal domain impairs in vitro SSA activity and reduces isochromosome formation in rad51∆ cells (Onaka et al, 2020), showing that Rad52-dependent recombination promotes isochromosome formation. At fission yeast centromeres, Rad51-dependent recombination predominates, and Rad52-dependent recombination is suppressed (Zafar et al, 2017).…”

“…Rad52 promotes gene conversion as well as GCRs in the absence of Rad51 (Onaka et al, 2020). However, Msh2-Msh3 and Mus81 are not required for the gene conversion branch of Rad52-dependent recombination.…”

“…Three types of GCRs have been detected using ChL C : translocation, isochromosome, and truncation, which can be distinguished by the length (Nakamura et al, 2008;Onaka et al, 2020;Onaka et al, 2016) (Figure 1A). Among them, isochromosomes are formed by recombination between inverted repeats at centromeres.…”

“…We have shown that Rad52 causes isochromosome formation in rad51∆ cells, using the rad52-R45K mutation that impairs SSA activity of Rad52 protein (Onaka et al, 2020). To determine 9 whether Rad8-dependent PCNA K107 ubiquitination plays a role in the Rad52-dependent GCR pathway, we introduced the rad8∆ or pcn1-K107R mutation into rad52-R45K rad51∆ cells ( Figure 4A).…”

“…Intriguingly, a mutation in DNA polymerase α (Pol α) required for lagging-strand synthesis increases Rad52-dependent recombination without changing the total rate of recombination at centromeres (Onaka et al, 2020), suggesting that the formation of ssDNA gaps during DNA replication might be a rate-limiting step of Rad52-dependent recombination. Rad52-dependent recombination leads not only GCRs but also gene conversion in the absence of Rad51.…”