Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

Results from a high-resolution deoxyribonuclease I (DNase I) footprinting titration procedure are described that identify preferred daunomycin binding sites within the 160 bp tyr T DNA fragment. We have obtained single-bond resolution at 65 of the 160 potential binding sites within the tyr T fragment and have examined the effect of 0-3.0 microM total daunomycin concentration on the susceptibility of these sites toward digestion by DNase I. Four types of behavior are observed: (i) protection from DNase I cleavage; (ii) protection, but only after reaching a critical total daunomycin concentration; (iii) enhanced cleavage; (iv) no effect of added drug. Ten sites were identified as the most strongly protected on the basis of the magnitude of the reduction of their digestion product band areas in the presence of daunomycin. These were identified as the preferred daunomycin binding sites. Seven of these 10 sites are found at the end of the triplet sequences 5'ATGC and 5'ATCG, where the notation AT indicates that either A or T may occupy the position. The remaining three strongly protected sites are found at the ends of the triplet sequence 5'ATCAT. Of the preferred daunomycin binding sites we identify in this study, the sequence 5'ATCG is consistent with the specificity predicted by the theoretical studies of Chen et al. [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466] and is the very sequence to which daunomycin is observed to be bound in two recent X-ray crystallographic studies. Solution studies, theoretical studies, and crystallographic studies have thus converged to provide a consistent and coherent picture of the sequence preference of this important anticancer antibiotic.

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.

Condensation of the chromatin fiber and transcriptional inhibition during mitosis is associated with the redistribution of many DNA-and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quantitative gel analysis with proteins extracted from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing negative charges in the NBD demonstrate that the negative charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the negative charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1 ؊/؊ cells indicates that the negatively charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.During mitosis the chromatin fiber is fully condensed and most of the transcriptional activity is inhibited (24, 34). These processes are associated with modifications in the tail of the core histones (11,18,23,62) and with changes in the location of DNA and chromatin binding proteins (31,41). Components of the transcriptional apparatus, transcription factors (35,50,51), and chromatin remodeling complexes (43, 54), are displaced from mitotic chromatin, while proteins that promote condensation seem to be enriched in this chromatin (14,26,32,58).Mitotic chromosomes are also depleted of high-mobility group B (HMGB) (HMG1 and -2) and HMGN (HMG14 and -17) (7,21,27,29,53) groups of proteins that serve as architectural elements and affect the structure and function of chromatin (5, 10). (Note that the nomenclature of the high-mobility-group proteins has been recently revised; HMGN1 was HMG-14, and HMGN2 was HMG-17. For a full description see the HMG Chromosomal Proteins Nomenclature Home Page http://www.informatics.jax.org/mgihome/nomen/genefamilies /hmgfamily.shtml.) HMGN proteins are the only nuclear proteins that specifically recognize the generic structure of the 147-bp nucleosome core particle (8). The proteins bind cooperatively to nucleosomes and form complexes containing two molecules of either HMGN1 or HMGN2 (49). Several types of experiments suggest that HMGN1 and HMGN2 proteins decompact chromatin and enhance transcription from chromatin, but not DNA, templates (reviewed in references 5 and 10). The proteins decompact the chromatin fiber by targeting two main elements known to compact chromatin: histone H1 (1, 19) and the amino terminus of histone H3 (60).The intrace...

Novel proteomics platforms, such as the aptamer‐based SOMAscan platform, can quantify large numbers of proteins efficiently and cost‐effectively and are rapidly growing in popularity. However, comparisons to conventional immunoassays remain underexplored, leaving investigators unsure when cross‐assay comparisons are appropriate. The correlation of results from immunoassays with relative protein quantification is explored by SOMAscan. For 63 proteins assessed in two chronic obstructive pulmonary disease (COPD) cohorts, subpopulations and intermediate outcome measures in COPD Study (SPIROMICS), and COPDGene, using myriad rules based medicine multiplex immunoassays and SOMAscan, Spearman correlation coefficients range from −0.13 to 0.97, with a median correlation coefficient of ≈0.5 and consistent results across cohorts. A similar range is observed for immunoassays in the population‐based Multi‐Ethnic Study of Atherosclerosis and for other assays in COPDGene and SPIROMICS. Comparisons of relative quantification from the antibody‐based Olink platform and SOMAscan in a small cohort of myocardial infarction patients also show a wide correlation range. Finally, cis pQTL data, mass spectrometry aptamer confirmation, and other publicly available data are integrated to assess relationships with observed correlations. Correlation between proteomics assays shows a wide range and should be carefully considered when comparing and meta‐analyzing proteomics data across assays and studies.

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.

Nucleolar protein B23 is a putative ribosome assembly factor with a high affinity for peptides containing nuclear localization signals (NLSs). The interactions of various NLS-containing peptides with two B23 isoforms (B23.1 and B23.2) were examined using equilibrium dialysis and Scatchard analyses. The KD for protein B23 binding to a peptide containing the SV40 T-antigen NLS sequence was approximately 1 microM with a stoichiometry of 1:1 (peptide:protein). No significant differences were seen between the two B23 isoforms in their affinities for any of the peptides tested. Binding by a reverse sequence SV40 T-NLS peptide showed a nonlinear Scatchard plot: this peptide was unable displace the correct sequence peptide, suggesting that the reverse sequence peptide binds to a different site on the protein. A peptide containing the sequence required for nucleolar localization of the HIV-1 Rev protein had an affinity for B23 approximately 10-fold greater than that of the SV40 T-NLS. However, with a sequence sufficient only for Rev location in the nucleoplasm, the affinity for B23 was diminished to a level between that of the longer Rev sequence and the SV40 T-NLS. In competition binding assays, the Rev NLS peptide was able to displace the SV40 T NLS, indicating that both peptides bind to the same site on protein B23. There was no detectable binding to protein B23 by a peptide containing the bipartite NLS of nucleoplasmin. Phosphorylation of protein B23 by casein kinase II enhanced its affinity for the SV40 T- and Rev-derived peptides approximately 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)