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.
Highlights
Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs)
Cells harbouring ChLC were grown in Edinburgh minimum medium supplemented with uracil and adenine (EMM + UA), and plated onto yeast nitrogen base (YNB) media: YNB + UA and YNB + UA + 5-fluoroorotic acid (5FOA), on which Leu+ and Leu+ Ura– colonies are formed, respectively
Contrary to GCR, rad52Δ and rad51Δ rad52Δ cells exhibited higher rates of chromosome loss than rad51Δ cells, indicating that Rad52-dependent single-strand annealing (SSA) plays a role in maintaining chromosomes in Rad51-deficient cells. These results demonstrate that Rad[52] is involved in GCRs that occur in the absence of Rad[51]
Summary
Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). The third pathway, called microhomology-mediated end joining (MMEJ) or alternative end joining[10], uses very short homologous DNA sequences and occurs independently of Rad[51] and Rad[52] In addition to their roles in DNA damage repair, an increasing body of evidence suggests that SSA and MMEJ are involved in tumorigenesis, in contrast to Rad51-dependent recombination[11,12,13,14]. We have shown that Rad[51] and Rad[54] promote non-crossover recombination between centromere repeats, thereby suppressing Mus81-dependent crossover recombination that results in isochromosome formation[26,27] It is unclear how homologous pairing occurs in the GCR event independently of Rad[51]. It is unknown how Rad51-dependent recombination predominates at centromeres
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