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

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 homo...

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Autores principales:	Jie Su, Ran Xu, Piyusha Mongia, Naoko Toyofuku, Takuro Nakagawa
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Genetics QH426-470
Acceso en línea:	https://doaj.org/article/89a007c032634961b84e6246ff9712ec
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spelling	oai:doaj.org-article:89a007c032634961b84e6246ff9712ec2021-12-02T20:02:55ZFission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.1553-73901553-740410.1371/journal.pgen.1009671https://doaj.org/article/89a007c032634961b84e6246ff9712ec2021-07-01T00:00:00Zhttps://doi.org/10.1371/journal.pgen.1009671https://doaj.org/toc/1553-7390https://doaj.org/toc/1553-7404Gross 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.Jie SuRan XuPiyusha MongiaNaoko ToyofukuTakuro NakagawaPublic Library of Science (PLoS)articleGeneticsQH426-470ENPLoS Genetics, Vol 17, Iss 7, p e1009671 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Genetics QH426-470
spellingShingle	Genetics QH426-470 Jie Su Ran Xu Piyusha Mongia Naoko Toyofuku Takuro Nakagawa Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.
description	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.
format	article
author	Jie Su Ran Xu Piyusha Mongia Naoko Toyofuku Takuro Nakagawa
author_facet	Jie Su Ran Xu Piyusha Mongia Naoko Toyofuku Takuro Nakagawa
author_sort	Jie Su
title	Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.
title_short	Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.
title_full	Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.
title_fullStr	Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.
title_full_unstemmed	Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination.
title_sort	fission yeast rad8/hltf facilitates rad52-dependent chromosomal rearrangements through pcna lysine 107 ubiquitination.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/89a007c032634961b84e6246ff9712ec
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_version_	1718375670647619584

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

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