Nucleolar protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase alpha activity

Phosphorylated retinoblastoma protein and nucleolar protein B23 are putative stimulatory factors for DNA polymerase alpha. We showed that these two factors interacted with each other and stimulated the activity of DNA polymerase alpha synergistically. B23 exists in two isoforms designated as B23.1 and B23.2. While B23.1 bound to a retinoblastoma protein-conjugated column, B23.2 did not. These results indicate that B23.1 can directly bind to retinoblastoma protein. It was also shown that B23 was co-immunoprecipitated with both retinoblastoma protein and DNA polymerase alpha from a HeLa cell extract by monoclonal antibodies raised against these components. These results suggest that these three proteins exist as a complex in cells, at least in part. The simultaneous addition of both B23.1 and retinoblastoma protein caused stimulation of DNA polymerase alpha activity that is much higher than the sum of the stimulation by retinoblastoma protein and B23.1 alone. The maximal stimulation was attained at the molar ratio of DNA polymerase alpha/retinoblastoma protein/B23.1 = 1:1:12. Since B23 exists as a hexamer in solution, it may act as a stimulator of DNA polymerase alpha in a form of double-hexamer, in concert with the phosphorylated retinoblastoma protein.     

The ribonuclease activity of nucleolar protein B23.

Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.     

State of oncomarker protein B23/nucleophosmin in HeLa cells

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38–40 kDa) and oligomers (210–230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210–230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.     

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35-40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C- and N- terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.     

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35–40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C-and N-terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.     

A single gene codes for two forms of rat nucleolar protein B23 mRNA

Protein B23 (38 kDa, pI = 5.1) is an abundant RNA-associated nucleolar phosphoprotein and putative ribosome assembly factor. A full length cDNA clone (lambda JH1) encoding a major expressed form of rat protein B23, now designated B23.1, was reported recently (Chang, J. H., Dumbar, T. S., and Olson, M. O. J. (1988) J. Biol. Chem. 263, 12824-12837). In this paper the isolation from a rat brain library and sequence of a cDNA clone (lambda JH2) coding for a second form (B23.2) of protein B23 is reported. Isoforms B23.1 and B23.2 are polypeptides of 292 and 257 amino acids, respectively. The 5'-untranslated regions of the two cDNAs and the amino-terminal 255 amino acids of the proteins are identical in the two isoforms. However, the 3'-untranslated regions of the mRNAs are completely different, and the dipeptide Gly-Gly in B23.1 (residues 256 and 257) is replaced by Ala-His in B23.2 indicating that the former is not a precursor of the latter. The finding of AGGT sequences in the 3' regions of lambda JH1 suggest the presence of intron-exon boundaries at the point where the two cDNAs begin to differ. To investigate the origin of the two isoforms, two rat genomic libraries were screened with oligonucleotide probes based on sequences from the unique regions of the two cDNAs. One of the genomic clones isolated (lambda JH125) contained a 6.5-kilobase fragment encoding the 3' end of both cDNAs. lambda JH125 contains four exons designated W, X, Y, and Z in the order indicated. Exons W and X encode 36 amino acids at the carboxyl terminus of B23.2, whereas exons W, Y, and Z encode the carboxyl-terminal 71 amino acid residues of B23.1. Exons X and Z each contain distinct 3'-untranslated sequences in which are found polyadenylation signals. These data suggest that two different mRNAs are formed by alternative splicing of separate 3' segments onto a common 5' region.     

Intrinsically disordered regions of nucleophosmin/B23 regulate its RNA binding activity through their inter- and intra-molecular association

Nucleophosmin (NPM1/B23) is a nucleolar protein implicated in growth-associated functions, in which the RNA binding activity of B23 plays essential roles in ribosome biogenesis. The C-terminal globular domain (CTD) of B23 has been believed to be the RNA binding domain because the splicing variant B23.2 lacking the CTD binds considerably less efficiently to RNA. However, the recognition of target RNAs by B23 remains poorly understood. Herein, we report a novel mechanism by which B23 recognizes specific RNA targets. We observed that the nucleolar retention of B23.3 lacking the basic region of B23.1 was lower than that of B23.1 because of its low RNA binding activity. Circular dichroism measurements indicated that the basic region and adjacent acidic regions of B23 are intrinsically disordered regions (IDRs). Biochemical analyses revealed that the basic IDR alone strongly binds to RNA with low specificity. The excessive RNA binding activity of the basic IDR was restrained by intra-molecular interaction with the acidic IDR of B23. Chemical cross-linking experiments and fluorescent labeling of bipartite tetracysteine-tagged proteins suggested that the inter- and intra-molecular interactions between the two IDRs contribute to the regulation of the RNA binding activity of CTD to control the cellular localization and functions of B23.     

Stimulation of DNA polymerase alpha activity by microtubule-associated proteins.

Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner. This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer. DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column. These events suggested that a physical binding occurred between the polymerase and MAP2. Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer. A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer. MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer. Product analyses showed that MAP2 did not significantly alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli. Other MAPs, tau and 190-kDa MAP, could substitute for MAP2. Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo.     

Studies on deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Variations of the enzyme activity during growth

1. RNA polymerase activity of Escherichia coli extracts prepared from cells in exponential and stationary phases of growth, when measured in the presence and absence of external template, showed significant qualitative differences. 2. In both extracts, polymerase activity was higher when assayed with external template, suggesting the presence of a pool of enzyme not bound to cellular DNA. 3. In the crude extract, the fraction of enzyme bound to cellular DNA is higher during the exponential phase of growth. 4. A method is described for the purification of enzyme molecules not tightly bound to cellular DNA from exponential- and stationary-phase cultures. 5. Purified enzyme preparations showed differences in template requirement and subunit composition. 6. On phosphocellulose chromatography of stationary-phase enzyme, a major portion of polymerase activity eluted from the column with 0.25m-KCl. In the case of exponential-phase enzyme, polymerase activity eluted from a phosphocellulose column mainly with 0.35m-KCl. 7. Enzyme assays done with excess of bacteriophage T(4) DNA showed a strong inhibition of stationary-phase enzyme by this template. The exponential-phase enzyme was only slightly inhibited by excess of bacteriophage T(4) DNA.     

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.     

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.     

The RNA binding activity of a ribosome biogenesis factor,nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.     

Hepatitis B virus: DNA polymerase activity of deletion mutants

The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. VII. Responsiveness is associated with expression of a product(s) of the V beta 8 gene family present on the T cell receptor alpha/beta for antigen

Mycoplasma arthritidis produces a potent soluble T lymphocyte mitogen (MAM) which is dependent upon accessory cells bearing the alpha-chain of the I-E molecule (E alpha). Lymphocytes from the RIIIS mouse strain which possess E alpha yet whose T cells fail to recognize the MAM-E alpha complex were shown not to express V beta 8.1, 8.2 or 8.3 gene products present on the TCR-alpha/beta by virtue of their lack of reactivity with the F23.1 mAb. Because lymphocytes from the congenic B10.RIII mouse strain reacted positively with F23.1, we examined the progeny from (RIIIS x B10.RIII)F1 x RIIIS backcross mice for cosegregation of lymphocytes expressing F23.1-reactive sites and ability to proliferate in response to MAM. Whereas lymphocytes of all mice responded to Con A, only lymphocytes from progeny expressing F23.1-reactive cells responded to MAM. Similar studies were conducted on progeny from the F23.1- SWR mouse which was backcrossed to (SWR x B10.RIII)F1 mice. Of the E alpha-bearing progeny, there was a direct correlation between lymphocyte expression of F23.1 determinants and ability to respond to MAM. These results established that MAM reactivity was dependent upon a product(s) of the V beta locus of the TCR-alpha/beta. Nodal and thymic lymphocytes cultured for 3 days in vitro with MAM exhibited clonal expansion of F23.1 and F23.2-reactive cells as compared with cultures treated with Con A. We also demonstrated that F23.1 and F23.2 mAb inhibited the ability of lymphocytes to proliferate in response to MAM but had little effect on responses to Con A. The combined data suggest that the MAM-E alpha complex can utilize a V beta 8 gene product(s) on the TCR-alpha/beta.     

Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis.

The Bacillus subtilis DNA-dependent RNA polymerase holoenzyme and core enzyme each contain approximately two atoms of zinc per molecule. When the dissociated subunits of the enzyme are passed through a blue dextran-Sepharose affinity column, only the beta subunit binds to the column. The total zinc content of the enzyme is tightly bound to the beta subunit. Dialysis studies suggest that the two zinc ions differ in the strength of their association with the beta subunit. The presence of zinc in beta is consistent with several other lines of evidence which indicate that this subunit is dirrectly involved in phosphodiester bond formation. The blue dextran-Sepharose column procedure should be useful in future studies of the dissociation and reassociation of the enzyme since the method is rapid and provides excellent recovery of the beta subunit as well as the alpha and beta' subunits of the RNA polymerase.     

On the binding of tRNA to Escherichia coli RNA polymerase.

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.     

Amino acid and nucleotide sequence homologies among E. coli RNA polymerase core enzyme subunits, DNA primase, elongation factor Tu, F1-ATPase alpha, ribosomal protein L3, DNA polymerase I, T7 phage DNA polymerase, and MS2 phage RNA replicase beta subunit

The 330 residue-long N-terminal domains (NTDs) of beta and beta' subunits of the Escherichia coli RNA polymerase (RPase) core enzyme were found to be significantly homologous to the entire length of its alpha subunit. The C-terminal domains (CTDs) of the RPase beta subunit and DNA primase (dnaG protein) were not only strongly homologous to each other but also considerably homologous to the RPase alpha, suggesting that an alpha subunit-like enzyme must have been commonly ancestral to core enzyme subunits and primase. The N-terminal region (NTR) of RPase alpha was also found to show significant homologies with NTRs of the E. coli EF-Tu and F1-ATPase alpha subunit, and a possible weak homology with ribosomal protein L3. A most important finding was that the C-terminal regions (CTRs) of DNA polymerase (DPase) I, T7 phage DPase and MS2 phage RNA replicase beta subunit are closely homologous with one another. These CTRs showed considerable homologies to RPase alpha NTD and RPase beta CTD. These conclusions are based on statistical evaluations of homologies in base and/or amino acid sequence alignments.     

Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.