Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum.

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH₂[³²P]O₄ and [³H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.     

DNA-binding proteins related to the dosage of specific yeast chromosomes

A comparison has been made of the $\text{in vitro}$ DNA-binding proteins of specific aneuploid and isogenic euploid cells of $\text{Saccharomyces cerevisiae}$ by DNA-cellulose chromatography. We have been able to detect changes in the level of a small fraction of the yeast DNA-binding proteins which can be related to the dosage of specific yeast chromosomes. At least four proteins show a dosage related to the cellular level of chromosome I and at least one protein shows a dosage related to the level of chromosome VI.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cmr1 protein preferentially binds to UV-damaged DNA <Emphasis Type="Italic">in vitro</Emphasis>

DNA metabolic processes such as DNA replication, recombination, and repair are fundamentally important for the maintenance of genome integrity and cell viability. Although a large number of proteins involved in these pathways have been extensively studied, many proteins still remain to be identified. In this study, we isolated DNA-binding proteins from Saccharomyces cerevisiae using DNA-cellulose columns. By analyzing the proteins using mass spectrometry, an uncharacterized protein, Cmr1/YDL156W, was identified. Cmr1 showed sequence homology to human Damaged-DNA binding protein 2 in its C-terminal WD40 repeats. Consistent with this finding, the purified recombinant Cmr1 protein was found to be intrinsically associated with DNA-binding activity and exhibited higher affinity to UV-damaged DNA substrates. Chromatin isolation experiments revealed that Cmr1 localized in both the chromatin and supernatant fractions, and the level of Cmr1 in the chromatin fraction increased when yeast cells were irradiated with UV. These results suggest that Cmr1 may be involved in DNA-damage responses in yeast.     

Mitochondrial DNA-binding proteins that bind preferentially to supercoiled molecules containing the D-loop region of Xenopus laevis mtDNA

From the bulk of the Xenopus laevis mitochondrial proteins insoluble in 1% Triton X-100 + 1M NaCl, we have isolated, by DNA-cellulose chromatography, a protein fraction enriched in DNA-binding proteins. This fraction contains proteins showing a specific affinity for supercoiled DNA molecules containing the mitochondrial DNA displacement-loop region, as measured by filter binding and competition assays.     

DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0–2.5 S, corresponding to a molecular weight of about 20 000–25 000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100–200 mM NaCl and is relatively independent of temperature and pH. MgCl₂ and MnCl₂ at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4·10^?11 M. There are at least 1·10⁵ DNA-binding protein molecules/HeLa cell.     

Finding nuclear localization signals

A variety of nuclear localization signals (NLSs) are experimentally known although only one motif was available for database searches through PROSITE. We initially collected a set of 91 experimentally verified NLSs from the literature. Through iterated ‘in silico mutagenesis’ we then extended the set to 214 potential NLSs. This final set matched in 43% of all known nuclear proteins and in no known non-nuclear protein. We estimated that >17% of all eukaryotic proteins may be imported into the nucleus. Finally, we found an overlap between the NLS and DNA-binding region for 90% of the proteins for which both the NLS and DNA-binding regions were known. Thus, evolution seemed to have used part of the existing DNA-binding mechanism when compartmentalizing DNA-binding proteins into the nucleus. However, only 56 of our 214 NLS motifs overlapped with DNA-binding regions. These 56 NLSs enabled a de novo prediction of partial DNA-binding regions for ~800 proteins in human, fly, worm and yeast.     

Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of k_exch = 6 × 10⁴ M⁻¹s⁻¹. The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.     

DNA-binding proteins of Xenopus laevis: synthesis during oogenesis-ovulation and early embryogenesis

DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with $\text{[}{}^3\text{H}\text{]}\text{tryptophan}$ and $\text{[}{}^{14}\text{C}\text{]}\text{lysine}$ indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium $\text{[}{}^{32}\text{P}\text{]}\text{phosphate}$. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.     

Analysis of epidermal proteins for DNA-binding activity

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.     

DNA-binding proteins present in varicella-zoster virus-infected cells.

DNA-binding proteins present in varicella-zoster virus-infected cells were identified by DNA-cellulose chromatography of radioactively labeled cell extracts. Seven virus-specific proteins, ranging in molecular weight from approximately 175,000 to 21,000, showed affinity for single- or double-stranded DNA or both. These proteins include the varicella-zoster virus major capsid protein, a phosphorylated tegument protein, and a 125,000-molecular-weight species which may be analogous to the major DNA-binding protein of herpes simplex virus. We also identified a number of DNA-binding phosphoproteins by these procedures. Finally, protein blot studies were carried out to determine whether these proteins bind preferentially to virus rather than to host cell DNA.     

Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity

SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure–function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5–Slx8 may act directly on DNA to promote genome stability.     

DNA-cellulose column chromatography of phosphorylated nucleolar nonhistone proteins

Summary A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [³²P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA.     

Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix

In our previous work we showed that DNaseI-like protein from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix retained its activity at salt concentrations as high as 4 M NaCl and the key factor allowing this was the C-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs. The further investigations revealed that this domain originated from proteins related to bacterial competence ComEA/ComE proteins. It is likely that in the course of evolution the DNA-binding domain from these proteins was fused to a metallo-β-lactamase superfamily domain. Very likely such domain organization having proteins subsequently “donated” the DNA-binding domain to bacterial DNases. In this study we have mimicked this evolutionary step by fusing bovine DNaseI and DNA-binding domains. We have created two fusions: one harboring the DNA-binding domain of DNaseI-like protein from Thioalkalivibrio sp. K90mix and the second one harboring the DNA-binding domain of bacterial competence protein ComEA from Bacillus subtilis. Both domains enhanced salt tolerance of DNaseI, albeit to different extent. Molecular modeling revealed the essential differences between their interaction with DNA shedding some light on the differences in salt tolerance. In this study we have enhanced salt tolerance of bovine DNaseI; thus, we successfully mimicked the Nature’s evolutionary engineering that created the extremely halotolerant bacterial DNase. We have demonstrated that the newly engineered DNaseI variants can be successfully used in applications where activity of the wild type bovine DNaseI is impeded by buffers used.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.     

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.     

Site-directed transposon integration in human cells

The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.     

Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays

The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579. Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells. In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells. Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped previously for dimerization by co-translation and immunoprecipitation in vitro. Comparison of the domains of the IE2 protein required for CRS binding and dimerization in yeast suggests that these activities correlate precisely with requirements for the negative autoregulation function of the IE2 protein in mammalian cells.     

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.