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PubMed2001

Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined. KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity. We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features. Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains. The Thumb domain of KOD DNA polymerase shows an "opened" conformation. The fingers subdomain possessed many basic residues at the side of the polymerase active site. The residues are considered to be accessible to the incoming dNTP by electrostatic interaction. A beta-hairpin motif (residues 242-249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69. Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments. The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity.

Amino Acid Sequence↗
PubMed1994

Ultrastructural localization of epidermal growth factor (EGF)-receptor transcripts in the cell nucleus using pre-embedding in situ hybridization in combination with ultra-small gold probes and silver enhancement.

A high-resolution in situ hybridization method is described for localizing epidermal growth factor (EGF)-receptor transcripts in nuclei of A431 epidermoid carcinoma cells. The method is based upon the use of ultra-small gold particles in combination with silver enhancement. The RNA of the EGF-receptor was detected mainly around the nucleoli. After removal of the DNA using nucleases and high salt extraction, the RNA of the EGF-receptor appears to be associated with the nuclear matrix. The RNA of the EGF-receptor was observed in close contact with the SC-35 splicing protein, but no exact colocalization was observed. These results demonstrate that high resolution pre-embedding in situ hybridization in combination with immunocytochemistry, both using ultra-small gold as a detection method, provides a powerful tool to unravel the organization of nuclear processes.

3T3 Cells↗
PubMed1997

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein.

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.

3T3 Cells↗
PubMed2003

Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system.

The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.

Base Sequence↗
PubMed2002

Immunohistological detection of BRAF25 in human prostate tumor and cancer specimens.

BRAF25 is an alternatively spliced protein of BRAF35 (see associated paper). We have mapped the BRAF25 gene to chromosome sub-band 19p13.3, a region where loss of chromosomal heterozygosity has been reported in about 50% of ovarian cancers. Because of the high incidence of genetic links of prostate cancer to breast and ovarian cancers, we investigated the BRAF25 expression in the prostate specimens. Immunohistochemical analysis using antibodies specific for BRAF25 revealed a strong immunostaining in sections of the benign prostatic hyperplasia (BPH). The staining was concentrated on the nuclei of cells facing the lumen of prostatic glands, even though the sporadic nuclei of cells in stromas were also stained. However, the expression of BRAF25 was dramatically reduced in intermediate prostate cancer and absent in advanced prostate cancer. Preincubation of the antibody with the immunizing peptide abolished immunostaining in BPH specimens. Therefore, the expression of BRAF25 was gradually lost in prostate cancer.

Antibodies↗
PubMed2006

A novel deletion mutation in CCM1 gene (krit1) is detected in a Chinese family with cerebral cavernous malformations.

Cerebral Cavernous Malformations (CCM) are vascular malformations that are mostly located in the central nervous system (CNS) and occasionally within the skin and retina, which are classified into three types (CCM1, CCM2 and CCM3) by being located at different loci on chromosomes. At present, CCM1 (7q21), CCM2 (7p13-p15) and CCM3 (3q25.2-q27) are respectively linked to krit1 (Krev interaction trapped gene 1), MGC4607 and PDCD10 (programmed cell death 10). In this work, we identified a novel "GTA" deletion mutation at the acceptor splicing site of intron9/exon10 on krit1. The mutation results in an abnormally spliced protein by creating a premature termination code at the 23rd amino acid downstream from the sequence alteration. Our results are consistent with previous research on krit1 mutations and confirm the conclusion that KRIT1 haploinsufficiency may be the underlying mechanism of CCM1.

Adult↗
PubMed2002

Insertion of a reversible redox switch into a rare-cutting DNA endonuclease.

Target sites for homing endonucleases occur infrequently in complex genomes. As a consequence, these enzymes can be used in mammalian systems to introduce double-strand breaks at recognition sites inserted within defined loci to study DNA repair by homologous and nonhomologous recombination. Using homing endonucleases for gene targeting in vivo would be more feasible if temporal or spatial regulation of their enzymatic activity were possible. Here, we show that the DNA cleavage activity of the yeast PI-SceI homing endonuclease can be turned on and off using a redox switch. Two cysteine pairs (Cys-64/Cys-344 and Cys-67/Cys-365) were separately inserted into flexible DNA binding loop(s) to create disulfide bonds that lock the endonuclease into a nonproductive conformation. The cleavage activities of the reduced Cys-64/Cys-344 and Cys-67/Cys-365 variants are similar or slightly lower than that of the control protein, but the activities of the proteins in the oxidized state are decreased more than 30-fold. Modulating the activity of the proteins is easily accomplished by adding or removing the reducing agent. We show that defects in DNA binding account for the decreased DNA cleavage activities of the proteins containing disulfide bonds. Interestingly, the Cys-67/Cys-365 variant toggles between two different DNA binding conformations under reducing and oxidizing conditions, which may permit the identification of structural differences between the two states. These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch.

Amino Acid Substitution↗
PubMed2005

Conformational heterogeneity at position U37 of an all-RNA hairpin ribozyme with implications for metal binding and the catalytic structure of the S-turn.

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.

Crystallization↗
PubMedDate not supplied

Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor.

To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a controllable self-splicing protein element in the form of a mini-intein was used to optimize the recovery of proteins for both batch and flow purification strategies. The ability to recover purified proteins was demonstrated using a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To characterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant dispersion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). Insight into the nature of the cleavage reaction and its regulation was obtained via temperature- and pH-dependent kinetic data.

Chromatography, Affinity↗
PubMed1998

Molecular evolution of viruses--Past and Present, Part 2--An introduction.

The evolution of viruses is reviewed within the perspective of the concepts on the evolution of the lipid membrane bound vesicular structures in the prebiotic soup through the ideas on evolution of cells during the RNA World and the transition into the DNA World. The ancient Archeae bacteria and their retrons that carry the bacterial reverse transcriptase gene and their unique protein splicing capability provide an indication of the evolutionary path for retroviruses and, independently, for RNA and DNA viruses of the prokaryotic Archeae bacteria and the eukaryotic yeast and fungi.

Archaea↗
PubMed2026

Analysis of structure and conservation for supporting functional evaluation of PMS2 missense variants.

Germline defects in mismatch repair (MMR) genes are known to significantly increase the risk of developing certain types of cancers, notably colorectal and endometrial cancers. These conditions are characterized under Lynch syndrome. Accurate diagnosis of this predisposition, along with meaningful predictive testing for family members, necessitates the identification of pathogenic variants. However, classifying small coding genetic variants identified in cancer patients is very challenging, specifically in the case of PMS2 variants, since PMS2 pathogenic variants display a lower penetrance and less severe phenotype and therefore a lower tumor burden in affected families. We have assembled clinical data on four PMS2 missense variants of uncertain significance (VUS) identified in 23 patients (p.(Asp286Gly), p.(Asn335Ser), p.(Ile679Thr) and p.(Arg799Trp)). For these variants, functional testing was performed (RNA splicing, protein stability and catalytic activity). Since many protein ortholog sequences and accurate predictive models from AlphaFold2 are available, we also included a systematic analysis of residue conservation and structural role (ConStruct assessment). Overall, our findings indicate that p.(Asp286Gly) and p.(Arg799Trp) behave similarly to wild-type PMS2 and are thus probably neutral. In contrast, p.(Asn335Ser) and p.(Ile679Thr) conferred defects in protein expression or MMR activity. These could be explained by the relevant roles of these amino acids in MLH1-PMS2-N-terminal dimerization (p.Asn335) and C-terminal dimerization (p.Ile679). Our data thus suggest that p.(Asp286Gly) and p.(Arg799Trp) are benign, while the tumor risk in the other two variants remains to be established. Taken together, we suggest roadmaps for the individualized evaluation of difficult uncertain variants by comprising information from all available sources.

Humans↗
PubMed1994

Cross-reactivity between autoimmune anti-U1 snRNP antibodies and neutralizing epitopes of HIV-1 gp120/41.

We report extensive amino acid sequence homology between HIV-1 gp120/41, and > 33% of a U1 RNA-associated splicing protein, 70K. The latter is a target of autoimmune anti-RNP antibodies in mixed connective tissue disease (MCTD). The homologies, involving dominant epitopes of 70K and neutralizing epitopes of gp120/41, are the basis for mutual antibody cross-reactivity. A key finding is that the epitope GRAFVTIG in the V3 loop of gp120 (strain IIIB) is homologous to the functionally essential U1 RNA-binding site of 70K. ELISA data reveal a mean reactivity of anti-RNP antibodies to V3 IIIB that is as high as that of HIV sera. V3 MN, containing the framework sequence G-AF-T, also cross-reacts with anti-RNP antibodies, as do hydrophilic epitopes in gp41 homologous to the COOH end of 70K. Further, there is strong cross-reactivity between HIV sera and 70K in Western blots. In contrast, antibodies from a related autoimmune disorder, Sjögren's syndrome (SS), are neither V3 nor gp41 selective. We conclude that the substantial cross-reactivities reported here are due to conserved, antigenically dominant B cell epitopes having homologous counterparts in 70K and gp120/41. Because antibody production in both MCTD and HIV-1 infection is T cell dependent, the results imply that common T cell clones are also activated in these two disease paradigms. Further exploration of the mechanisms that activate these clones, and that control their divergent fates in MCTD and AIDS, may provide new insights into immune dysregulation in HIV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

The thy pol-2 intein of Thermococcus hydrothermalis is an isoschizomer of PI-TliI and PI-TfuII endonucleases.

THY Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same allelic family as TLI Pol-2 (PI-TLII), Tfu Pol-2 (PI-TFUII) and TspTY Pol-3 mini-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intein was cloned, expressed in Escherichia coli and purified. The intein is a specific endonuclease (PI-THY:I) which cleaves the inteinless sequence of the THY DNA pol gene. Moreover, PI-TLII, PI-TFUII and PI-THYI are very similar endonucleases which cleave DNA in the same optimal conditions at 70 degrees C yielding similar 3'-hydroxyl overhangs of 4 bp and the reaction is subject to product inhibition. The three enzymes are able to cleave the three DNA sequences spanning the pol-c site and a 24 bp consensus cleavage site was defined for the three isoschizomers. However, the exact size of the minimal cleavage site depends both on the substrate sequence and the endonuclease. The inability of the isoschizomers to cleave the inteinless DNA polymerase gene from Pyrococcus spp. KOD is due to point substitutions on the 5' side of the pol-c site, suggesting that the absence of inteins of this allelic family in DNA polymerase genes from Pyrococcus spp. can be linked to small differences in the target site sequence.

Alleles↗