Abstract
Gross chromosomal rearrangements (GCRs), including translocation, deletion, and inversion, can cause cell death and genetic diseases such as cancer in multicellular organisms. Rad51, a DNA strand exchange protein, suppresses GCRs by repairing spontaneous DNA damage through a conservative way of homologous recombination, gene conversion. On the other hand, Rad52 that catalyzes single-strand annealing (SSA) causes GCRs using homologous sequences. However, the detailed mechanism of Rad52-dependent GCRs remains unclear. Here, we provide genetic evidence that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. In rad51Δ cells, loss of Rad8 eliminated 75% of the isochromosomes resulting from centromere inverted repeat recombination, showing that Rad8 is essential for the formation of the majority of isochromosomes in rad51Δ cells. Rad8 HIRAN and RING finger mutations reduced GCRs, suggesting that Rad8 facilitates GCRs through 3’ DNA-end binding and ubiquitin ligase activity. Mms2 and Ubc4 but not Ubc13 ubiquitin-conjugating enzymes were required for GCRs. Consistent with this, mutating PCNA K107 rather than the well-studied PCNA K164 reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. In contrast to GCRs, PCNA K107R did not significantly change gene conversion rates, suggesting a specific role of PCNA K107 ubiquitination in GCRs. PCNA K107R enhanced temperature-sensitive growth defects of DNA ligase I cdc17-K42 mutant, implying that PCNA K107 ubiquitination occurs when Okazaki fragment maturation fails. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 and Rad8 ubiquitin ligase for GCRs. These data suggest that Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs by changing the PCNA clamp structure.
Highlights
Faithful repair of DNA damage, such as DNA double-strand breaks (DSBs), is critical to maintaining genome integrity [1,2]
To detect Leu+ Ura− Ade− Gross chromosomal rearrangements (GCRs) clones that have lost both ura4+ and ade6+, cells were grown in Edinburgh minimum media supplemented with uracil and adenine (EMM+UA) and plated on FOA+UA media containing 5-fluoroorotic acid (5-FOA) which is toxic to ura4+ cells
Leu+ Ura− colonies formed on FOA +UA plates were transferred to Edinburgh minimal medium (EMM)+U to inspect adenine auxotrophy
Summary
Faithful repair of DNA damage, such as DNA double-strand breaks (DSBs), is critical to maintaining genome integrity [1,2]. Homologous recombination is considered an error-free DSB repair mechanism, as it uses intact DNA as the template. When accompanied by crossover or break-induced replication, nonallelic homologous recombination between repetitive sequences in a genome results in gross chromosomal rearrangements (GCRs), including translocation, deletion, and inversion [3,4]. Isochromosomes, whose arms mirror each other, are the GCR products formed by recombination between inverted repeats around centromeres [5]. Replication fork stalling can cause GCR events without DSB formation [6]. Replication forks reassembled by homologous recombination following fork stalling are prone to switch template strands and form acentric and dicentric isochromosomes using nearby inverted repeats [7].
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