A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing.

We describe an in vitro protocol for quickly generating overlapping terminal-labeled restriction fragments for DNA sequence analysis via the Maxam-Gilbert technique. The protocol involves introducing mercurated nucleotides into one end of a region to be sequenced, partial digestion with several restriction enzymes and terminal-labeling, separation of the mercurated restriction enzymes and terminal-labeling, separation of the mercurated restriction fragments from non-mercurated ones on a thiol column and resolution of the different mercurated fragments on one preparative agarose gel. The protocol was used to determine the nucleotide sequence of a 980 base pair cDNA that contains the coding region for a variable surface glycoprotein of Trypanosoma brucei. It could just as quickly and easily be used to obtain many terminal-labeled overlapping restriction fragments covering a region of several kilobases.     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Interaction of 6-thiopurines and thiol-containing RNA with a cellulose mercurial.

Thiol-containing bases such as 6-mercaptopurine bind to mercurated cellulose under conditions where non-thiol bases do not bind. Both RNA and thiol-containing RNA bind to mercurated cellulose, but the affinity for thiol-containing RNA is considerably greater. Ligands such as CN^? and $\text{S}{}_2\text{O}{}_3{}^{\mathrm{2?}}$ inhibit this binding to mercurated cellulose, and conditions can be found which permit almost complete retention, of thiol-containing RNA with low retention of non-thiol-containing RNA. A simple method is described for separating newly synthesised RNA containing thiol groups from older RNA which does not contain thiol groups.     

Synthesis and distribution of primer RNA in nuclei of CCRF-CEM leukemia cells

The distribution of primer RNA and RNA-primed nascent DNA in nuclei of CCRF-CEM leukemia cells was examined, and the primer RNA purified from the nuclear matrices of these cells was characterized. RNA-primed nascent DNA was radiolabeled by incubating whole-cell lysates with [alpha-32P]ATP and [3H]dTTP in the presence of approximately physiological concentrations of the remaining ribo- and deoxyribonucleoside triphosphates. The primer RNA was purified by cesium chloride density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. Nuclear subfractionation studies revealed that at least 94% of the primer RNA and RNA-primed nascent DNA were located within the insoluble matrix fraction of the nucleus. The predominant primer RNA isolated from the nuclear matrix was 8-10 nucleotides in length, and several lines of evidence indicated that this oligoribonucleotide was the functional primer RNA. Essentially all of the matrix primer RNA was covalently linked to the newly replicated DNA as demonstrated by its buoyant density in cesium chloride gradients, phosphate-transfer analysis, and sensitivity to DNase I. Analysis of 32P transfer from [alpha-32P]dTTP revealed a random distribution of ribonucleotides at the 3'-end of the primer RNA. Data obtained from mixing experiments indicated that the association of RNA-primed nascent DNA with the nuclear matrix was not the result of aggregation of these fragments with the nuclear matrix. No significant amount of either primer RNA, RNA-primed nascent DNA, or phosphate transfer was detected in the high-salt-soluble (nonmatrix) fraction of the nucleus, although the nonmatrix fraction contained most of the newly replicated DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Using RNase sequence specificity to refine the identification of RNA-protein binding regions

Massively parallel pyrosequencing is a high-throughput technology that can sequence hundreds of thousands of DNA/RNA fragments in a single experiment. Combining it with immunoprecipitation-based biochemical assays, such as cross-linking immunoprecipitation (CLIP), provides a genome-wide method to detect the sites at which proteins bind DNA or RNA. In a CLIP-pyrosequencing experiment, the resolutions of the detected protein binding regions are partially determined by the length of the detected RNA fragments (CLIP amplicons) after trimming by RNase digestion. The lengths of these fragments usually range from 50-70 nucleotides. Many genomic regions are marked by multiple RNA fragments. In this paper, we report an empirical approach to refine the localization of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion. We present two regions to which multiple amplicons map as examples to demonstrate this approach.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro.

In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.     

Alkali-labile structures linked to the 5'' ends of Bacillus subtilis short DNA chains.

Alkali-labile portion covalently linked to the 5' ends of Bacillus subtilis short DNA chains, the putative primer RNA for discontinuous DNA synthesis, was isolated and analyzed using a temperature sensitive DNA polymerase I mutant, which accumulates nascent DNA fragments at a restrictive temperature. A novel oligonucleotide structure as well as mono- to triribonucleotide stretches were isolated at the 5' end of the short DNA chains. A structure for the novel oligonucleotide is proposed to be p5' X3' pp5' rN, where X represents unidentified nucleoside with a peculiar property. Possible metabolic relationship between these molecules and primer RNA has been discussed.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

Homologous RNA polymerase binding to Escherichia coli DNA: a study of the distribution of stable binding sites.

The number and the distribution of the sites of Escherichia coli DNA that form stable complexes with the homologous RNA polymerase (class A sites according to Hinkle and Chamberlin [3]) have been investigated. Almost all the DNA can bind RNA polymerase, even when fragmented at short (undergenic) size; this general non-promoter-specific binding is highly labile and is not temperature-dependent. The range of RNA polymerase/DNA ratios that give rise to the stable temperature-dependent complexes was examined. The amount and the distribution of class A complexes were studied analysing the dissociation of complexes formed by RNA polymerase on DNA fragments of various length. The E. coli genome appears to form 3.8 X 10(3) stable complexes; the majority of these complexes shows a short-range distribution (800-1200 base pairs). The rest is more widely spaced (1200-6000 base pairs).     

New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

Influence of fragment size on DNA quantitation using DNA-binding proteins and a sensor-based analytical system: applications in the testing of biological products.

A novel immunoassay system which rapidly quantifies picogram levels of total DNA was characterized with respect to the effects of DNA length. Nine chromatographically purified HaeIII restriction fragments of phi X174 were tested. Assay performance was found to be dependent on both the amount and length of DNA present in the sample. DNA fragments longer than 100 base pairs (bp) could be quantitatively detected with this system. Fragments shorter than 100 bp inhibited assay performance and thus could be detected through the use of inhibition studies; however, only qualitative information could be obtained. DNA fragments approximately 10 nucleotides in length had no apparent effect on assay performance. The size of the binding site (number of bases) required for each DNA-binding protein to bind to a nucleic acid fragment is suggested as an explanation for the observed influence of DNA size on assay performance. The total DNA assay was used in conjunction with a Pharmacia FPLC system to characterize the size distribution and amount of DNA in two partially purified biopharmaceutical samples. The results indicate that the majority of residual DNA in these samples is less than 600 bp in length. This technique can be used to rapidly determine the DNA size distribution in an in-process or final product biopharmaceutical sample. This data can then be used in process design and optimization for removal of residual DNA in biological products.     

The reactions of mercurated pyrimidine nucleotides with thiols and with hydrogen sulfide.

In the presence of thiols, 5-mercuripyrimidine nucleotides are quantitatively converted to 5-thiomercuri derivatives, but these compounds are unstable and decompose at a rate dependent on the nature of the thiol. The decomposition involves three different reactions and proceeds via a symmetrical mercury derivative of the nucleotide. The end product is the unmodified nucleotide. Similar reactions occur in the presence of hydrogen sulfide. Since mercurated nucleoside triphosphates are substrates for RNA- and DNA polymerase only in the form of thiomercuri derivatives, this implies that when DNA is replicated or transcribed in vitro with a mercurated substrate, the latter is rapidly demercurated to the unmodified substrate which is incorporated as well. Hence the product of the in vitro synthesis can only be partially mercurated in any one pyrimidine. Also, formation of cross-links in the resulting polymer is possible.