Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions.

ABSTRACT: Naked and protein-blocked DNA ends occur naturally during immune cell development, meiosis, and at telomeres as well as from aborted topoisomerase reactions, collapsed replication forks, and other stressors. Damaged DNA ends are dangerous in cells and if left unrepaired can lead to genomic rearrangement, loss of genetic information, and eventually cancer. Mre11 is part of the Mre11-Rad50-Nbs1 complex that recognizes DNA double-strand breaks and has exonuclease and endonuclease activities that help to initiate the repair processes to resolve these broken DNA ends. In fact, these activities are crucial for proper DNA damage repair pathway choice. Here, using Pyrococcus furiosus Mre11, we question how two Mre11 separation-of-function mutants, one previously described but the second first described here, maintain endonuclease activity in the absence of exonuclease activity. To start, we performed solution-state NMR experiments to assign the side-chain methyl groups of the 64-kDa Mre11 nuclease and capping domains, which allowed us to describe the structural differences between Mre11 bound to exo- and endonuclease substrates. Then, through biochemical and biophysical characterization, including NMR structural and dynamics studies, we compared the two mutants and determined that both affect the dynamic features and double-stranded DNA binding properties of Mre11, but in different ways. In total, our results illuminate the structural and dynamic landscape of Mre11 nuclease function.

SUBMITTER: Rahman S

PROVIDER: S-EPMC7231638 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function.

Project description:The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.

| S-EPMC4418825 | biostudies-literature

Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast.

Project description:Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

| S-EPMC5705816 | biostudies-other

Perturbing cohesin dynamics drives MRE11 nuclease-dependent replication fork slowing.

Project description:Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.

| S-EPMC6379725 | biostudies-literature

The Universally Conserved Residues Are Not Universally Required for Stable Protein Expression or Functions of Cryptochromes.

Project description:Universally conserved residues (UCRs) are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. Cryptochromes are photoreceptors in plants or light-independent components of the circadian clocks in mammals. We experimentally analyzed 51 UCRs of Arabidopsis cryptochrome 2 (CRY2) that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, we found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one photoresponses analyzed. Our finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.

| S-EPMC8204711 | biostudies-literature

Physiological protein blocks direct the Mre11-Rad50-Xrs2 and Sae2 nuclease complex to initiate DNA end resection.

Project description:DNA double-strand break repair by homologous recombination is initiated by DNA end resection, which is commenced by the Mre11-Rad50-Xrs2 complex and Sae2 in yeast. Here we report that the nonhomologous end joining factor Ku limits the exonuclease activity of Mre11 and promotes its endonuclease to cleave 5'-terminated DNA strands at break sites. Following initial endonucleolytic cleavage past the obstacle, Exo1 specifically extends the resection track, leading to the generation of long 3' overhangs that are required for homologous recombination. These experiments provide mechanistic insights into how short-range and long-range DNA end resection enzymes overcome obstacles near broken DNA ends to initiate recombination.

| S-EPMC5795779 | biostudies-literature

Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.

Project description:Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.

| S-EPMC15089 | biostudies-literature

CCRXP: exploring clusters of conserved residues in protein structures.

Project description:Conserved residues forming tightly packed clusters have been shown to be energy hot spots in both protein-protein and protein-DNA complexes. A number of analyses on these clusters of conserved residues (CCRs) have been reported, all pointing to a crucial role that these clusters play in protein function, especially protein-protein and protein-DNA interactions. However, currently there is no publicly available tool to automatically detect such clusters. Here, we present a web server that takes a coordinate file in PDB format as input and automatically executes all the steps to identify CCRs in protein structures. In addition, it calculates the structural properties of each residue and of the CCRs. We also present statistics to show that CCRs, determined by these procedures, are significantly enriched in 'hot spots' in protein-protein and protein-RNA complexes, which supplements our more detailed similar results on protein-DNA complexes. We expect that CCRXP web server will be useful in studies of protein structures and their interactions and selecting mutagenesis targets. The web server can be accessed at http://ccrxp.netasa.org.

| S-EPMC2896124 | biostudies-literature

Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Project description:Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

| S-EPMC4256157 | biostudies-literature

Restricted mobility of conserved residues in protein-protein interfaces in molecular simulations.

Project description:Conserved residues in protein-protein interfaces correlate with residue hot-spots. To obtain insight into their roles, we have studied their mobility. We have performed 39 explicit solvent simulations of 15 complexes and their monomers, with the interfaces varying in size, shape, and function. The dynamic behavior of conserved residues in unbound monomers illustrates significantly lower flexibility as compared to their environment, suggesting that already before binding they are constrained in a boundlike configuration. To understand this behavior, we have analyzed the inter- and intrachain hydrogen-bond residence-time in the interfaces. We find that conserved residues are not involved significantly in hydrogen bonds across the interface as compared to nonconserved. However, the monomer simulations reveal that conserved residues contribute dominantly to hydrogen-bond formation before binding. Packing of conserved residues across the trajectories is significantly higher before and after the binding, rationalizing their lower mobility. Backbone torsional angle distributions show that conserved residues assume restricted regions of space and the most visited conformations in the bound and unbound trajectories are similar, suggesting that conserved residues are preorganized. Combined with previous studies, we conclude that conserved residues, hot spots, anchor, and interface-buried residues may be similar residues, fulfilling similar roles.

| S-EPMC2292389 | biostudies-literature

Nuclease deficiencies alter plasma cell-free DNA methylation profiles.

Project description:The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

| S-EPMC8559716 | 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