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 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.

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

10.1101/2021.01.05.425428 ◽

2021 ◽

Author(s):

Jie Su ◽

Naoko Toyofuku ◽

Takuro Nakagawa

Keyword(s):

Fission Yeast ◽

Ubiquitin Ligase ◽

Inverted Repeat ◽

Chromosomal Rearrangements ◽

Ring Finger ◽

Sliding Clamp ◽

Gross Chromosomal Rearrangements ◽

Ubiquitin Ligase Activity

AbstractRad52 recombinase can cause gross chromosomal rearrangements (GCRs). However, the mechanism of Rad52-dependent GCRs remains unclear. Here, we show that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. Loss of Rad8 reduced isochromosomes resulting from centromere inverted repeat recombination. 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 proteins were required for GCRs. Consistent with this, PCNA K107R but not K164R mutation greatly reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 ubiquitination for GCRs. This study uncovers the role of Rad8-dependent PCNA K107 ubiquitination in Rad52-dependent GCRs.

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Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast

The EMBO Journal ◽

10.1038/emboj.2009.265 ◽

2009 ◽

Vol 28 (21) ◽

pp. 3400-3412 ◽

Cited By ~ 32

Author(s):

Helen Tinline-Purvis ◽

Andrew P Savory ◽

Jason K Cullen ◽

Anoushka Davé ◽

Jennifer Moss ◽

...

Keyword(s):

Fission Yeast ◽

Gene Conversion ◽

Chromosomal Rearrangements

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Mechanisms underlying genome instability mediated by formation of foldback inversions in Saccharomyces cerevisiae

eLife ◽

10.7554/elife.58223 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Bin-zhong Li ◽

Christopher D Putnam ◽

Richard David Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Instability ◽

Genetic Diseases ◽

Chromosomal Rearrangements ◽

Genetic System ◽

Dna Hairpins ◽

Large Loop ◽

Gross Chromosomal Rearrangements ◽

Single Strand Annealing ◽

Strand Annealing

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

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Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

Molecular and Cellular Biology ◽

10.1128/mcb.00524-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 897-906 ◽

Cited By ~ 21

Author(s):

Thomas J. Pohl ◽

Jac A. Nickoloff

Keyword(s):

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Single Strand ◽

Homology Search ◽

Wild Type ◽

Dsb Repair ◽

Single Strand Annealing ◽

In The Wild ◽

Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.103 ◽

2001 ◽

Vol 157 (1) ◽

pp. 103-118 ◽

Cited By ~ 16

Author(s):

Janet R Mullen ◽

Vivek Kaliraman ◽

Samer S Ibrahim ◽

Steven J Brill

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Stability ◽

Protein Complexes ◽

Ring Finger ◽

Dna Helicase ◽

Open Reading Frames ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Helicases ◽

Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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An E3 Ubiquitin Ligase RNF139 Serves as a Tumor-Suppressor in Glioma

Journal of Molecular Neuroscience ◽

10.1007/s12031-021-01860-4 ◽

2021 ◽

Author(s):

Xiaofeng Chen ◽

Weiping Kuang ◽

Yong Zhu ◽

Bin Zhou ◽

Xiaosong Li ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Lines ◽

Ubiquitin Ligase ◽

Glioma Cell ◽

Protein Modification ◽

E3 Ubiquitin Ligase ◽

Ring Finger ◽

Glioma Cells ◽

Migration And Invasion ◽

Tissue Samples

AbstractGlioma is highly lethal because of its high malignancy. Ubiquitination, a type of ubiquitin-dependent protein modification, has been reported to play an oncogenic or tumor-suppressive role in glioma development, depending on the targets. Ring finger protein 139 (RNF139) is a membrane-bound E3 ubiquitin ligase serving as a tumor suppressor by ubiquitylation-dependently suppressing cell growth. Herein, we firstly confirmed the abnormal downregulation of RNF139 in glioma tissues and cell lines. In glioma cells, ectopic RNF139 overexpression could inhibit, whereas RNF139 knockdown could aggravate the aggressive behaviors of glioma cells, including hyperproliferation, migration, and invasion. Moreover, in two glioma cell lines, RNF139 overexpression inhibited, whereas RNF139 knockdown enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT serine/threonine kinase 1 (AKT). In a word, we demonstrate the aberration in RNF139 expression in glioma tissue samples and cell lines. RNF139 serves as a tumor-suppressor in glioma by inhibiting glioma cell proliferation, migration, and invasion and promoting glioma cell apoptosis through regulating PI3K/AKT signaling.

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Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.6891-6899.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 6891-6899 ◽

Cited By ~ 92

Author(s):

Xuan Wang ◽

Grzegorz Ira ◽

José Antonio Tercero ◽

Allyson M. Holmes ◽

John F. X. Diffley ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Temperature Sensitive ◽

Replication Proteins ◽

Dna Ligases ◽

Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

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Normal Immune System Development in Mice Lacking the Deltex-1 RING Finger Domain

Molecular and Cellular Biology ◽

10.1128/mcb.25.4.1437-1445.2005 ◽

2005 ◽

Vol 25 (4) ◽

pp. 1437-1445 ◽

Cited By ~ 15

Author(s):

Sébastien Storck ◽

Frédéric Delbos ◽

Nicolas Stadler ◽

Catherine Thirion-Delalande ◽

Florence Bernex ◽

...

Keyword(s):

B Cell ◽

Notch Signaling ◽

Ubiquitin Ligase ◽

Cell Lineage ◽

Ring Finger ◽

Mouse Development ◽

Ring Finger Domain ◽

Ubiquitin Ligase Activity ◽

Immune System Development ◽

Cell Commitment

ABSTRACT The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1Δ/Δ mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.

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