Yeast Ortholog Research Articles

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot-Marie-Tooth disease model.

Aminoacyl-tRNA synthetases are essential enzymes responsible for charging amino acids onto cognate tRNAs during protein synthesis. In histidyl-tRNA synthetase (HARS), autosomal dominant mutations V133F, V155G, Y330C and S356N in the HARS catalytic domain cause Charcot-Marie-Tooth disease type 2W (CMT2W), while tRNA-binding domain mutation Y454S causes recessive Usher syndrome type IIIB. In a yeast model, all human HARS variants complemented a genomic deletion of the yeast ortholog HTS1 at high expression levels. CMT2W associated mutations, but not Y454S, resulted in reduced growth. We show mistranslation of histidine to glutamine and threonine in V155G and S356N but not Y330C mutants in yeast. Mistranslating V155G and S356N mutants lead to accumulation of insoluble proteins, which was rescued by histidine. Mutants V133F and Y330C showed the most significant growth defect and decreased HARS abundance in cells. Here, histidine supplementation led to insoluble protein aggregation and further reduced viability, indicating histidine toxicity associated with these mutants. V133F proteins displayed reduced thermal stability in vitro, which was rescued by tRNA. Our data will inform future treatment options for HARS patients, where histidine supplementation may either have a toxic or compensating effect depending on the nature of the causative HARS variant.

Delineating transitions during the evolution of specialised peroxisomes: Glycosome formation in kinetoplastid and diplonemid protists.

One peculiarity of protists belonging to classes Kinetoplastea and Diplonemea within the phylum Euglenozoa is compartmentalisation of most glycolytic enzymes within peroxisomes that are hence called glycosomes. This pathway is not sequestered in peroxisomes of the third Euglenozoan class, Euglenida. Previous analysis of well-studied kinetoplastids, the ‘TriTryps’ parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., identified within glycosomes other metabolic processes usually not present in peroxisomes. In addition, trypanosomatid peroxins, i.e. proteins involved in biogenesis of these organelles, are divergent from human and yeast orthologues. In recent years, genomes, transcriptomes and proteomes for a variety of euglenozoans have become available. Here, we track the possible evolution of glycosomes by querying these databases, as well as the genome of Naegleria gruberi, a non-euglenozoan, which belongs to the same protist supergroup Discoba. We searched for orthologues of TriTryps proteins involved in glycosomal metabolism and biogenesis. Predicted cellular location(s) of each metabolic enzyme identified was inferred from presence or absence of peroxisomal-targeting signals. Combined with a survey of relevant literature, we refine extensively our previously postulated hypothesis about glycosome evolution. The data agree glycolysis was compartmentalised in a common ancestor of the kinetoplastids and diplonemids, yet additionally indicates most other processes found in glycosomes of extant trypanosomatids, but not in peroxisomes of other eukaryotes were either sequestered in this ancestor or shortly after separation of the two lineages. In contrast, peroxin divergence is evident in all euglenozoans. Following their gain of pathway complexity, subsequent evolution of peroxisome/glycosome function is complex. We hypothesize compartmentalisation in glycosomes of glycolytic enzymes, their cofactors and subsequently other metabolic enzymes provided selective advantage to kinetoplastids and diplonemids during their evolution in changing marine environments. We contend two specific properties derived from the ancestral peroxisomes were key: existence of nonselective pores for small solutes and the possibility of high turnover by pexophagy. Critically, such pores and pexophagy are characterised in extant trypanosomatids. Increasing amenability of free-living kinetoplastids and recently isolated diplonemids to experimental study means our hypothesis and interpretation of bioinformatic data are suited to experimental interrogation.

Computer-aided molecular modeling and structural analysis of the human centromere protein–HIKM complex

BackgroundProtein–peptide and protein–protein interactions play an essential role in different functional and structural cellular organizational aspects. While Cryo-EM and X-ray crystallography generate the most complete structural characterization, most biological interactions exist in biomolecular complexes that are neither compliant nor responsive to direct experimental analysis. The development of computational docking approaches is therefore necessary. This starts from component protein structures to the prediction of their complexes, preferentially with precision close to complex structures generated by X-ray crystallography.ResultsTo guarantee faithful chromosomal segregation, there must be a proper assembling of the kinetochore (a protein complex with multiple subunits) at the centromere during the process of cell division. As an important member of the inner kinetochore, defects in any of the subunits making up the CENP-HIKM complex lead to kinetochore dysfunction and an eventual chromosomal mis-segregation and cell death. Previous studies in an attempt to understand the assembly and mechanism devised by the CENP-HIKM in promoting the functionality of the kinetochore have reconstituted the protein complex from different organisms including fungi and yeast. Here, we present a detailed computational model of the physical interactions that exist between each component of the human CENP-HIKM, while validating each modeled structure using orthologs with existing crystal structures from the protein data bank.ConclusionsResults from this study substantiate the existing hypothesis that the human CENP-HIK complex shares a similar architecture with its fungal and yeast orthologs, and likewise validate the binding mode of CENP-M to the C-terminus of the human CENP-I based on existing experimental reports.Graphical abstract

Functional analysis of missense DARS2 variants in siblings with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation.

Biallelic pathogenic variants in the nuclear gene DARS2 (MIM# 610956), encoding the mitochondrial enzyme aspartyl-tRNA synthetase (MT-ASPRS) cause leukoencephalopathy with Brain Stem and Spinal Cord Involvement and Lactate Elevation (LBSL) (MIM# 611105), a neurometabolic disorder characterized by progressive ataxia, spasticity, developmental arrest or regression and characteristic brain MRI findings. Most patients exhibit a slowly progressive disease course with motor deterirartion that begins in childhood or adolescence, but can also occasionaly occur in adulthood. More severe LBSL presentations with atypical brain MRI findings have been recently described. Baker's yeast orthologue of DARS2, MSD1, is required for growth on oxidative carbon sources. A yeast with MSD1 knockout (msd1Δ) demonstrated a complete lack of oxidative growth which could be rescued by wild-type MSD1 but not MSD1 with pathogenic variants. Here we reported two siblings who exhibited developmental regression and ataxia with different age of onset and phenotypic severity. Exome sequencing revealed 2 compound heterozygous missense variants in DARS2: c.473A>T (p.Glu158Val) and c.829G>A (p.Glu277Lys); this variant combination has not been previously reported. The msd1Δ yeast transformed with plasmids expressing p.Glu259Lys, equivalent to human p.Glu277Lys, showed complete loss of oxidative growth and oxygen consumption, while the strain carrying p.Gln137Val, equivalent to human p.Glu158Val, showed a significant reduction of oxidative growth, but a residual ability to grow was retained. Structural analysis indicated that p.Glu158Val may interfere with protein binding of tRNAAsp, while p.Glu277Lys may impact both homodimerization and catalysis of MT-ASPRS. Our data illustrate the utility of yeast model and in silico analysis to determine pathogenicity of DARS2 variants, expand the genotypic spectrum and suggest intrafamilial variability in LBSL.

SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells.

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone-fold domain (HFD)-containing proteins forming three histone-fold (HF) pairs, to which the double HFD-containing SUPT3H adds one HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that (i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H, (ii) SUPT3H is not essential for mESC survival, but required for their growth and self-renewal, and (iii) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.

Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis.

Ribosome biogenesis is a fundamental and highly orchestrated process that involves hundreds of ribosome biogenesis factors. Despite advances that have been made in yeast, the molecular mechanism of ribosome biogenesis remains largely unknown in plants. We uncovered a WD40 protein, Shrunken and Embryo Defective Kernel 1 (SHREK1), and showed that it plays a crucial role in ribosome biogenesis and kernel development in maize (Zea mays). The shrek1 mutant shows an aborted embryo and underdeveloped endosperm and embryo-lethal in maize. SHREK1 localizes mainly to the nucleolus and accumulates to high levels in the seed. Depleting SHREK1 perturbs pre-rRNA processing and causes imbalanced profiles of mature rRNA and ribosome. The expression pattern of ribosomal-related genes is significantly altered in shrek1. Like its yeast (Saccharomyces cerevisiae) ortholog Periodic tryptophan protein 1 (PWP1), SHREK1 physically interacts with ribosomal protein ZmRPL7a, a transient component of the PWP1-subcomplex involved in pre-rRNA processing in yeast. Additionally, SHREK1 may assist in the A3 cleavage of the pre-rRNA in maize by interacting with the nucleolar protein ZmPOP4, a maize homolog of the yeast RNase mitochondrial RNA-processing complex subunit. Overall, our work demonstrates a vital role of SHREK1 in pre-60S ribosome maturation, and reveals that impaired ribosome function accounts for the embryo lethality in shrek1.

Keystone Malaria Symposium 2022: a vibrant discussion of progress made and challenges ahead from drug discovery to treatment

Keystone Malaria Symposium 2022: a vibrant discussion of progress made and challenges ahead from drug discovery to treatment

Full humanization of the glycolytic pathway in Saccharomyces cerevisiae.

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models.

Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast.

Fission yeast RNA polymerase II consists of 12 subunits, Rpb1-Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow-growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further gain an understanding of its physiological roles, in the present study we have elucidated the role of the Rpb9 subunit in chronological aging using fission yeast as the model organism. Our results provide evidence that the absence of Rpb9 reduces the chronological life span in fission yeast. Our data further shows that lack of Rpb9 in fission yeast causes oxidative stress sensitivity and accumulation of reactive oxygen species during the stationary phase. Our domain mapping experiments have demonstrated that the Rpb9 region encompassing its amino-terminal zinc finger domain and the central linker region is important for the role of Rpb9 in chronological aging. Finally, we also show that expression of the budding yeast or human Rpb9 ortholog can functionally complement the reduced chronological life span phenotype of the fission yeast rpb9 deletion mutant. Taken together, our study has identified a new role of the Rpb9 subunit in chronological aging.

Characterization of anti-porcine CXCL10 monoclonal antibodies

Abstract C-X-C motif chemokine ligand 10 (CXCL10) facilitates chemoattraction of important immune cells to tissues. As part of the USDA-NIFA Swine Immune Toolkit Initiative, our goal is to provide the veterinary community with new commercial immune reagents and standardized assays for future research efforts. For the chemokine target CXCL10, we used yeast expressed, recombinant porcine CXCL10 (rPoCXCL10) to generate a panel of α-CXCL10 monoclonal antibodies (mAbs). Each of the α-PoCXCL10 mAbs was assessed by ELISA using cross-inhibition analyses of biotinylated mAbs, and direct binding to orthologous yeast expressed CXCL10 proteins. As a result, we assigned six distinct epitope groups for the nine generated α-CXCL10 mAbs. Subsequently, we screened AF647-tagged α-CXCL10 mAbs for intracellular staining of pig immune cells using different stimulation conditions. Of the nine mAbs only two detected intracellular CXCL10 expression in PMA/ionomycin or rPoIFNγ-stimulated porcine cells. Further, cell characterization assays verified CXCL10+ cells as CD3-CD4-CD172+, with occasional CD4+ subsets. Overall, we have determined that α-CXCL10-1.4 mAb is the best mAb clone for intracellular staining and recommend it for use in analysis of intracellular signaling to detect interactions that regulate cell migration. A sandwich ELISA was also developed to quantitate CXCL10 protein expression; it verified reactivity with native porcine CXCL10. Overall, these tools will be useful for evaluating immunity against swine infectious diseases and host-responses to vaccines. New reagents identified by the Swine Immune Toolkit Initiative will undoubtedly advance future swine research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815. Supported by USDA-ARS project # 8042?32000?102 and USDA-NIFA AFRI grant # 2019-67015-29815

"Flexible hinge" dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy.

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on similar timescales as one-dimensional (1D) diffusion of proteins on DNA if effective in stalling these proteins as they scan DNA. We examined the flexibility and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC). A previous Forster resonance energy transfer (FRET) study mapped DNA conformational distributions with cytosine analog FRET pair primarily sensitive to DNA twisting/unwinding deformations (Chakraborty et al. Nucleic Acids Res. 46: 1240-1255 (2018)). These studies revealed B-DNA conformations for nonspecific (matched) constructs but significant unwinding for mismatched constructs specifically recognized by Rad4, even in the absence of Rad4. The timescales of these unwinding fluctuations, however, remained elusive. Here, we labeled DNA with Atto550/Atto647N FRET dyes suitable for fluorescence correlation spectroscopy (FCS). With these probes, we detected higher FRET in specific, mismatched DNA compared with matched DNA, reaffirming unwinding/bending deformations in mismatched DNA. FCS unveiled the dynamics of these spontaneous deformations at ~ 300µs with no fluctuations detected for matched DNA within the ~ 600ns-10ms FCS time window. These studies are the first to visualize anomalous unwinding/bending fluctuations in mismatched DNA on timescales that overlap with the < 500µs "stepping" times of repair proteins on DNA. Such "flexible hinge" dynamics at lesion sites could arrest a diffusing protein to facilitate damage interrogation and recognition.

Recipes for Inducing Cold Denaturation in an Otherwise Stable Protein.

Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the freezing point of water. Understanding the mechanisms that determine cold denaturation and the rules that permit its observation is an important challenge. A way to approach them is to be able to induce cold denaturation in an otherwise stable protein by means of mutations. Here, we studied CyaY, a relatively stable bacterial protein with no detectable cold denaturation and a high melting temperature of 54 °C. We have characterized for years the yeast orthologue of CyaY, Yfh1, a protein that undergoes cold and heat denaturation at 5 and 35 °C, respectively. We demonstrate that, by transferring to CyaY the lessons learnt from Yfh1, we can induce cold denaturation by introducing a restricted number of carefully designed mutations aimed at destabilizing the overall fold and inducing electrostatic frustration. We used molecular dynamics simulations to rationalize our findings and demonstrate the individual effects observed experimentally with the various mutants. Our results constitute the first example of rationally designed cold denaturation and demonstrate the importance of electrostatic frustration on the mechanism of cold denaturation.

The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer’s disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42‐mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co‐purification of Abeta42 with mitochondria and prevents Abeta42‐induced mitochondria‐dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax.

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochromec (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cytcrelease, closely resembling the natural Bax function in the cellular context. This regulated cell death process wasreverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.

SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones.

The histone chaperone complex FACT comprises SPT16 and SSRP1 and contributes to DNA replication, transcription, and repair, but how it plays such various roles is unclear. Here, we show that human SPT16 is ubiquitylated at lysine-674 (K674) by the DCAF14-CRL4 ubiquitin ligase. K674 is located in the middle domain of SPT16, and the corresponding residue of the yeast ortholog is critical for binding to histone H3.1-H4. We show that the middle domain of human SPT16 binds to histone H3.1-H4 and that this binding is inhibited by K674 ubiquitylation. Cells with heterozygous knockin of a K674R mutant of SPT16 manifest reduction of both SPT16 ubiquitylation and H3.1 in chromatin, a reduced population in mid S phase, impaired proliferation, and increased susceptibility to S phase stress. Our data thus indicate that SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones and may thereby control DNA replication-coupled histone incorporation into chromatin.

Histone Deacetylases as Modulators of the Crosstalk Between Skeletal Muscle and Other Organs.

Skeletal muscle plays a major role in controlling body mass and metabolism: it is the most abundant tissue of the body and a major source of humoral factors; in addition, it is primarily responsible for glucose uptake and storage, as well as for protein metabolism. Muscle acts as a metabolic hub, in a crosstalk with other organs and tissues, such as the liver, the brain, and fat tissue. Cytokines, adipokines, and myokines are pivotal mediators of such crosstalk. Many of these circulating factors modulate histone deacetylase (HDAC) expression and/or activity. HDACs form a numerous family of enzymes, divided into four classes based on their homology to their orthologs in yeast. Eleven family members are considered classic HDACs, with a highly conserved deacetylase domain, and fall into Classes I, II, and IV, while class III members are named Sirtuins and are structurally and mechanistically distinct from the members of the other classes. HDACs are key regulators of skeletal muscle metabolism, both in physiological conditions and following metabolic stress, participating in the highly dynamic adaptative responses of the muscle to external stimuli. In turn, HDAC expression and activity are closely regulated by the metabolic demands of the skeletal muscle. For instance, NAD+ levels link Class III (Sirtuin) enzymatic activity to the energy status of the cell, and starvation or exercise affect Class II HDAC stability and intracellular localization. SUMOylation or phosphorylation of Class II HDACs are modulated by circulating factors, thus establishing a bidirectional link between HDAC activity and endocrine, paracrine, and autocrine factors. Indeed, besides being targets of adipo-myokines, HDACs affect the synthesis of myokines by skeletal muscle, altering the composition of the humoral milieu and ultimately contributing to the muscle functioning as an endocrine organ. In this review, we discuss recent findings on the interplay between HDACs and circulating factors, in relation to skeletal muscle metabolism and its adaptative response to energy demand. We believe that enhancing knowledge on the specific functions of HDACs may have clinical implications leading to the use of improved HDAC inhibitors for the treatment of metabolic syndromes or aging.

The Structure of the Arabidopsis PEX4-PEX22 Peroxin Complex-Insights Into Ubiquitination at the Peroxisomal Membrane.

Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported. Here we report the X-ray crystal structure of the PEX4-PEX22 peroxin complex from the reference plant Arabidopsis thaliana. PEX4 is a ubiquitin-conjugating enzyme (UBC) that ubiquitinates proteins associated with the peroxisomal membrane, and PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. We co-expressed Arabidopsis PEX4 as a translational fusion with the soluble PEX4-interacting domain of PEX22 in E. coli. The fusion was linked via a protease recognition site, allowing us to separate PEX4 and PEX22 following purification and solve the structure of the complex. We compared the structure of the PEX4-PEX22 complex to the previously published structures of yeast orthologs. Arabidopsis PEX4 displays the typical UBC structure expected from its sequence. Although Arabidopsis PEX22 lacks notable sequence identity to yeast PEX22, it maintains a similar Rossmann fold-like structure. Several salt bridges are positioned to contribute to the specificity of PEX22 for PEX4 versus other Arabidopsis UBCs, and the long unstructured PEX22 tether would allow PEX4-mediated ubiquitination of distant peroxisomal membrane targets without dissociation from PEX22. The Arabidopsis PEX4-PEX22 structure also revealed that the residue altered in pex4-1 (P123L), a mutant previously isolated via a forward-genetic screen for peroxisomal dysfunction, is near the active site cysteine of PEX4. We demonstrated in vitro UBC activity for the PEX4-PEX22 complex and found that the pex4-1 enzyme has reduced in vitro ubiquitin-conjugating activity and altered specificity compared to PEX4. Our findings illuminate the role of PEX4 and PEX22 in peroxisome structure and function and provide tools for future exploration of ubiquitination at the peroxisome surface.

“Flexible hinge” dynamics in mismatched DNA uncovered with laser temperature-jump and fluorescence correlation spectroscopy

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on <500-µs timescale if effective in stalling proteins as they scan DNA. Here, we measured conformational distributions and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC) that recognizes diverse lesions from UV-damage or other genotoxins and initiates DNA repair.

Refining the domain architecture model of the replication origin firing factor Treslin/TICRR.

Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan origin firing regulation hub Treslin/TICRR and its yeast orthologue Sld3 share the Sld3-Treslin domain and the adjacent TopBP1/Dpb11 interaction domain. We report a revised domain architecture model of Treslin/TICRR. Protein sequence analyses uncovered a conserved Ku70-homologous β-barrel fold in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, Sld3-Treslin domain, and TopBP1/Dpb11 interaction domain, flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C terminus. The CIT includes a von Willebrand factor type A domain. Unexpectedly, MTBP, Treslin/TICRR, and Ku70/80 share the same N-terminal domain architecture, von Willebrand factor type A and Ku70-like β-barrels, suggesting a common ancestry. Binding experiments using mutants and the Sld3-Sld7 dimer structure suggest that the Treslin/Sld3 and MTBP/Sld7 β-barrels engage in homotypic interactions, reminiscent of Ku70-Ku80 dimerization. Cells expressing Treslin/TICRR domain mutants indicate that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during metazoan origin firing.

Expression Characterization of AtPDI11 and Functional Analysis of AtPDI11 D Domain in Oxidative Protein Folding.

The formation and isomerization of disulfide bonds mediated by protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) is of fundamental importance in eukaryotes. Canonical PDI structure comprises four domains with the order of a-b-b′-a′. In Arabidopsis thaliana, the PDI-S subgroup contains only one member, AtPDI11, with an a-a′-D organization, which has no orthologs in mammals or yeast. However, the expression pattern of AtPDI11 and the functioning mechanism of AtPDI11 D domain are currently unclear. In this work, we found that PDI-S is evolutionarily conserved between land plants and algal organisms. AtPDI11 is expressed in various tissues and its induction by ER stress is disrupted in bzip28/60 and ire1a/b mutants that are null mutants of key components in the unfolded protein response (UPR) signal transduction pathway, suggesting that the induction of AtPDI11 by ER stress is mediated by the UPR signaling pathway. Furthermore, enzymatic activity assays and genetic evidence showed that the D domain is crucially important for the activities of AtPDI11. Overall, this work will help to further understand the working mechanism of AtPDI11 in catalyzing disulfide formation in plants.