An antigenic region of topoisomerase I in DNA polymerase chain reaction-generated fragments recognized by autoantibodies of scleroderma patients.

Topoisomerase cDNA and various fragments thereof generated by the DNA polymerase chain reaction were cloned into plasmid expression vectors (pET series) and the expressed polypeptides were probed with scleroderma sera from seven different patients immunoreactive with topoisomerase I. All sera reacted selectively with a region between amino acid residues 405 and 484 of human topoisomerase I. This conclusion is based on loss of reactivity when this region was omitted from larger pieces. Other portions of topoisomerase I were not reactive with these autoantibodies. At least two different epitopes appear to be recognized within this region by different sera based on differences in immunoreactivity of the 405-484 region when expressed as C-terminal, N-terminal or internally within a peptide.

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations.

The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.

Identification of a phosphatase activity, toward the phosphopeptide pyroGlu-Asp-Asp-Ser(p)-Asp-Glu-Glu-Asn, in nuclear extract from HL-60 promyelocitic leukaemia cells.

Total protein extract from HL-60 cells was found to be able to dephosphorylate the RNA polymerase II octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn previously phosphorylated with protein kinase CKII (pCKII). Fractionation in cytoplasm, nuclear and chromatin extracts shows the phosphatase activity to be localized only in the nucleus, but not to be bound to the chromatin.

Functional domains of nucleolar phosphoprotein p120.

Nucleolar phosphoprotein p120 is a low abundance, proliferation-associated protein. Several functional domains have been characterized and are discussed here such as the antigenic domain recognized by a monoclonal antibody, the nuclear/nucleolar localization domain, phosphorylation domains of casein kinase II (CKII) and protein kinase C, a putative methylation domain and an RNA binding region. By sucrose gradient sedimentation analyses, protein p120 was shown to rapidly sediment with 60-80 S pre-rRNP particles but sedimented more slowly when treated with RNAse or salt suggesting binding to RNA. Nucleolar protein p120 differed from other nucleolar proteins such as C23 (nucleolin) and B23 (nucleophosmin) which sedimented more slowly near the top of the gradient.

Synthetic model peptides phosphorylated in vitro by NII kinase and cation-mediated interactions with DNA.

Minimum substrate requirements for nuclear NII kinase (casein II kinase) were analyzed with synthetic peptides modeled according to amino acid composition of phosphopeptides isolated from chromatin. Uncharged blocked peptide termini decreased the requirement for acidic clusters neighboring the phosphate acceptor amino acid (serine) such that only one group immediately N-terminal to serine was sufficient for kinase activity. Studies on peptide interaction with DNA showed that the model phosphopeptides bound to DNA only in the phosphorylated form suggesting involvement of phosphorylation in protein-DNA interactions yet to be identified.

Phosphorylation of human topoisomerase I by protein kinase C in vitro and in phorbol 12-myristate 13-acetate-activated HL-60 promyelocytic leukaemia cells.

Topoisomerase I was phosphorylated in vitro by protein kinase C (PKC) purified from rat brain with high affinity (Km about 0.1 microM). Tryptic phosphopeptide mapping indicated that two major topoisomerase I peptides phosphorylated in vivo were comigrating with minor peptides phosphorylated by PKC in vitro. Topoisomerase I phosphorylation was stimulated 3-fold in HL-60 cells exposed to the tumour promoter phorbol 12-myristate 13-acetate. The results suggest that topoisomerase I phosphorylation in HL-60 cells is indirectly controlled by PKC.

Binding of small phosphorylated chromatin peptides to DNA.

Low-molecular-weight peptides involved in gene expression and cell growth have been isolated from DNA preparation from eukaryotic cells. After phosphorylation with protein kinase CKII (pCKII) these peptides are able to bind to DNA in presence of divalent cations and salt/ethanol. This finding may explain the mechanism by which the peptides exert their activity.

Phosphorylation of the synthetic octapeptide pyroGlu-ASP-ASP-SER-ASP-GLU-GLU-ASN and binding to DNA in presence of divalent cations.

Small acidic peptides involved in gene expression have been isolated from prokaryotic and eukaryotic cells. Synthetic peptides, designed on the basis of native peptides characteristics, show a biological activity similar to that of native peptides in in vitro reconstituted systems. These synthetic peptides are able to bind to DNA in presence of divalent cations (Cu2+, Fe2+, Mg2+) and salt/ethanol.

Human topoisomerase I is phosphorylated in vitro on its amino terminal domain by protein kinase NII.

Topoisomerase I purified from HeLa cells was phosphorylated in vitro with protein kinase NII (pkNII) purified from calf thymus: this phosphorylation was inhibited by heparin. A peptide containing a sequence corresponding to a putative pkNII phosphorylation site in topoisomerase I was synthesized and phosphorylated with pkNII. HPLC and two-dimensional analysis show identity between the synthetic phosphorylated peptide and one topoisomerase I phosphopeptide indicating Ser10 as one of the in vitro pkNII phosphorylation sites in topoisomerase I.

Phosphopeptides derived from in vitro phosphorylated E. coli RNA polymerase bind to DNA and affect DNA transcription.

E. Coli RNA polymerase was phosphorylated with protein kinase CKII and allowed to bind to pBR322. After digestion of the RNA polymerase-pBR322 complex with proteinase K, the phosphopeptides that remained bound to DNA were extracted and analyzed. These phosphopeptides are able to bind again to DNA and to inhibit transcription.

Differential scanning calorimetry of chromatin at different levels of condensation.

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 degrees C, while the other two occur at 63 degrees C and 74 degrees C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 degrees C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.

Small acidic peptides are bound to E. coli DNA.

Low molecular weight peptides have been isolated by alkali extraction from deproteinized DNA of E. coli cells grown in the presence of radioactive glutamic acid or orthophosphate. The labeled peptides, purified by gel filtration chromatography on Sephadex G25 and G10, contain prevailingly glutamic acid, aspartic acid, glycine, serine and alanine. Electrophoretic studies at different pH show that some peptide fractions contain a phosphoric residue. The N-terminus of the phosphorylated peptides is apparently blocked and they were able to bind to DNA in the presence of Mg2+ ions. Moreover the acidic peptides extracted from E. coli DNA show a sharp activity in the control of lambda phage DNA transcription 'in vitro'.

In vitro phosphorylation of proteins tightly bound to DNA by protein kinase NII.

1. Highly purified DNA from calf thymus was phosphorylated with protein kinase NII. 2. Digestion with proteinase K of this DNA demonstrates proteins as phosphorylated component. 3. Gel filtration chromatography on Bio-Gel A-0.5m gel column shows a major protein peak between 50 and 70 kDa. 4. SDS gel electrophoresis, after hydrolysis, to digest completely DNA, shows three major phosphorylated bands corresponding to polypeptides of M(r) between 31 and 21 kDa. 5. After high voltage electrophoresis on TLC plates tryptic digested polypeptides show very similar phosphopeptides patterns.

Molecular models of acidic peptides from pea bud chromatin and seminal plasma. Divalent cations-mediated interaction with DNA.

Small acidic peptides have been isolated from biological fluids (blood and seminal plasma) and from chromatin of several tissues. Their biological activity is related to the control of cell growth and gene expression. This work is an approach to the study of peptide structure-function relationship. Purified fractions from seminal plasma and pea bud chromatin were subjected to fast ion bombardment mass spectrometry. The results obtained were analyzed according to biochemical characteristics of the peptides studied and some possible molecular models have been designed. Two of the proposed sequences were synthesized and their biological activity assayed in cells and cell-free systems. The results demonstrate that the synthetic peptides are able to bind to DNA in the presence of divalent cations (Mg2+, Fe2+, Cu2+) with consequent inhibition of DNA transcription.