Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mutations of the Yku80 C terminus and Xrs2 FHA domain specifically block yeast nonhomologous end joining.

ABSTRACT: The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic alpha-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.

SUBMITTER: Palmbos PL

PROVIDER: S-EPMC1316971 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Yeast Tdp1 regulates the fidelity of nonhomologous end joining.

Project description:Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5' extensions showed a high frequency of misrepair in Deltatdp1 cells. The joining errors in Deltatdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3' extensions or blunt ends did not show enhanced frequencies of errors, although Deltatdp1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3' nucleosidase activity regulates the processing of DNA ends by generating a 3' phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3' phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3' phosphate is a key step in Tdp1-mediated error prevention during NHEJ.

| S-EPMC2840102 | biostudies-literature

Positive selection of yeast nonhomologous end-joining genes and a retrotransposon conflict hypothesis.

Project description:Transposable elements have clearly played a major role in shaping both the size and organization of eukaryotic genomes. However, the evolution of essential genes in core biological processes may also have been shaped by coevolution with these elements. This would be predicted to occur in instances where host proteins are either hijacked for use by mobile elements or recruited to defend against them. To detect such cases, we have used the Saccharomyces cerevisiae-Saccharomyces paradoxus sibling species pair to identify genes that have evolved under positive selection. We identify 72 such genes, which participate in a variety of biological processes but are enriched for genes involved in meiosis and DNA repair by nonhomologous end-joining (NHEJ). We confirm the signature of positive selection acting on NHEJ genes using orthologous sequences from all seven Saccharomyces sensu stricto species. Previous studies have found altered rates of Ty retrotransposition when these NHEJ genes are disrupted. We propose that the evolution of these repair proteins is likely to have been shaped by their interactions with Ty elements. Antagonistic pleiotropy, where critical genes like those involved in DNA repair are also subject to selective pressures imposed by mobile elements, could favor alleles that might be otherwise deleterious for their normal roles related to genome stability.

| S-EPMC1693795 | biostudies-literature

Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast.

Project description:Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 3' flaps that can form during imprecise pairing. Indeed, a mutation in the 3'-5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ.

| S-EPMC2312331 | biostudies-literature

Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 complex to a double-strand break requires interactions with Yku80 and the Xrs2 FHA domain.

Project description:Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70-Yku80 (Ku), Dnl4-Lif1-Nej1 (DNA ligase IV), and Mre11-Rad50-Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2-Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2-Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80-Dnl4 and Xrs2-Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs-Lif1 and Yku80-Dnl4 interactions are important for formation of a productive ligase-DSB intermediate.

| S-EPMC2600923 | biostudies-literature

Rap1 prevents telomere fusions by nonhomologous end joining.

Project description:Telomeres protect chromosomes from end-to-end fusions. In yeast Saccharomyces cerevisiae, the protein Rap1 directly binds telomeric DNA. Here, we use a new conditional allele of RAP1 and show that Rap1 loss results in frequent fusions between telomeres. Analysis of the fusion point with restriction enzymes indicates that fusions occur between telomeres of near wild-type length. Telomere fusions are not observed in cells lacking factors required for nonhomologous end joining (NHEJ), including Lig4 (ligase IV), KU and the Mre11 complex. SAE2 and TEL1 do not affect the frequency of fusions. Together, these results show that Rap1 is essential to block NHEJ between telomeres. Since the presence of Rap1 at telomeres has been conserved through evolution, the establishment of NHEJ suppression by Rap1 could be universal.

| S-EPMC1201357 | biostudies-literature

DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining.

Project description:Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends.

| S-EPMC5649367 | biostudies-literature

The minimal Bacillus subtilis nonhomologous end joining repair machinery.

Project description:It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ) system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD) and Ku (BsuKu) proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i) inherent polymerization activity showing preferential insertion of NMPs, ii) specific recognition of the phosphate group at the downstream 5' end, iii) intrinsic ligase activity, iv) ability to promote realignments of the template and primer strands during elongation of mispaired 3' ends, and v) it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments.

| S-EPMC3656841 | biostudies-literature

Developmental modulation of nonhomologous end joining in Caenorhabditis elegans.

Project description:Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.

| S-EPMC1526663 | biostudies-literature

Nonhomologous end joining: a good solution for bad ends.

Project description:Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy.

| S-EPMC4024359 | biostudies-literature

Genome-wide screens for sensitivity to ionizing radiation identify the fission yeast nonhomologous end joining factor Xrc4.

Project description:Nonhomologous end joining (NHEJ) is the main means for repairing DNA double-strand breaks (DSBs) in human cells. Molecular understanding of NHEJ has benefited from analyses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In human cells, the DNA ligation reaction of the classical NHEJ pathway is carried out by a protein complex composed of DNA ligase IV (LigIV) and XRCC4. In S. cerevisiae, this reaction is catalyzed by a homologous complex composed of Dnl4 and Lif1. Intriguingly, no homolog of XRCC4 has been found in S. pombe, raising the possibility that such a factor may not always be required for classical NHEJ. Here, through screening the ionizing radiation (IR) sensitivity phenotype of a genome-wide fission yeast deletion collection in both the vegetative growth state and the spore state, we identify Xrc4, a highly divergent homolog of human XRCC4. Like other fission yeast NHEJ factors, Xrc4 is critically important for IR resistance of spores, in which no homologous recombination templates are available. Using both extrachromosomal and chromosomal DSB repair assays, we show that Xrc4 is essential for classical NHEJ. Exogenously expressed Xrc4 colocalizes with the LigIV homolog Lig4 at the chromatin region of the nucleus in a mutually dependent manner. Furthermore, like their human counterparts, Xrc4 and Lig4 interact with each other and this interaction requires the inter-BRCT linker and the second BRCT domain of Lig4. Our discovery of Xrc4 suggests that an XRCC4 family protein is universally required for classical NHEJ in eukaryotes.

| S-EPMC4455778 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data