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B M Alberts

Publications and source records attributed to B M Alberts.

At least 91 records · Page 5Linked to original sources
PubMed1983

Properties of the T4 bacteriophage DNA replication apparatus: the T4 dda DNA helicase is required to pass a bound RNA polymerase molecule.

The interaction of DNA replication forks with both stationary and transcribing RNA polymerase molecules has been examined in vitro, using the multienzyme T4 bacteriophage DNA replication system and purified E. coli RNA polymerase. We have found that a single stationary RNA polymerase molecule can block the movement of the T4 replication fork when bound to a promoter on a double-stranded fd DNA template. When transcription is allowed (in the same direction as replication), the replication fork appears to follow the moving RNA polymerase molecule at the relatively slow rate of transcription. The barriers to fork movement formed by E. coli RNA polymerase are eliminated by the addition of small amounts of a purified T4-encoded DNA helicase, the product of the dda gene. We find that replication complexes containing the dda protein cause stationary RNA polymerase molecules to dissociate from the DNA.

Adenosine Triphosphatases↗
PubMed1983

Affinity purification of bacteriophage T4 proteins essential for DNA replication and genetic recombination.

The bacteriophage T4 helix-destabilizing protein, the product of gene 32, has been immobilized on an agarose matrix and used for affinity chromatography of lysates of T4-infected Escherichia coli cells. At least 10 T4-encoded early proteins and 3 or 4 host proteins are specifically retained by this gene 32 protein column. Nine of the T4 proteins have been identified as being involved in either DNA replication or genetic recombination. Notably, the T4 DNA polymerase (gene 43 protein) and two major proteins in the recombination pathway (the products of genes uvsX and uvsY) are specifically bound. On a preparative scale, the column is useful for purification of the bound proteins.

Bacterial Proteins↗
PubMed1983

Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis.

Using differential interference contrast optics, combined with cinematography, we have studied the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm. We have supplemented these studies with data collected from fixed, stained, whole embryos. The following information has been obtained. The average duration of nuclear cycles 10, 11, 12 and 13 is about 9, 10, 12 and 21 min, respectively (25 degrees C). In these four cycles, the duration of that portion of the mitotic period that lacks a discrete nuclear envelope is 3, 3, 3 and 5 min, respectively. The length of nuclear cycle 14 varies in a position-specific manner throughout the embryo, the shortest cycles being of 65 min duration. During nuclear cycles 10 through 13, it is commonly observed in living embryos that the syncytial blastoderm nuclei enter (and leave) mitosis in one of two waves that originate nearly simultaneously from the opposite anterior and posterior poles of the embryo, and terminate in its midregion. From our preparations of quick-frozen embryos, we estimate that these mitotic waves take on average about half a minute to travel over the embryonic surface from pole to equator. The yolk nuclei, which remain in the core of the embryo when the rest of the nuclei migrate to the periphery, divide in synchrony with the migrating nuclei at nuclear cycles 8 and 9, and just after the now peripherally located nuclei at nuclear cycle 10. After cycle 10, these yolk nuclei cease dividing and become polyploid. The syncytial embryo has at least three distinct levels of cytoskeletal organization: structured domains of cytoplasm are organized around each blastoderm nucleus; radially directed tracks orient colchicine-sensitive saltatory transport throughout the peripheral cytoplasm; and a long-range organization of the core of the embryo makes possible coherent movements of the large inner yolk mass in concert with each nuclear cycle. This highly organized cytoplasm may be involved in providing positional information for the important process of nuclear determination that is known to occur during these stages.

Animals↗
PubMed1982

Inhibition of histone acetylation by N-[2-(S-coenzyme A)acetyl] spermidine amide, a multisubstrate analog.

A multisubstrate analog, formed by joining coenzyme A with spermidine through an acetic acid linkage, serves as a strong inhibitor (Ki less than 10(-8) M) of the acetylation of spermidine and histones by histone acetylase purified from calf thymus. In free solutions, this analog inhibited acetylation of the various nuclear histones to a similar extent. In isolated nuclei, this analog was found to inhibit acetylation of histones H2a and H2b very much more strongly than that of histones H3 and H4.

Acetylation↗
PubMed1982

Accessibility of newly synthesized chromatin to histone acetylase.

Pulse-chase experiments with [3H]lysine-labeled tissue culture cells reveal that newly synthesized nucleosomal histones H2B, H3, H4 (and possibly H2A) in chromatin are more accessible to histone acetylase in vivo than are older, pre-existing histones. Thus, when rat hepatoma cells are first pulse-labeled and then incubated in medium containing n-butyrate which blocks histone deacetylation, these newly synthesized histones become acetylated to a far greater extent than do their older homologues. As judged by its increased susceptibility to acetylation, the new chromatin matures at a surprisingly slow rate, the estimated half-time for maturation being about 35 min. Based on this data, we suggest that newly synthesized chromatin is in a relatively extended, accessible conformation, and that it slowly returns to a more compact conformation as it matures.

Acetyltransferases↗
PubMed1981

Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template.

We have recently developed an in vitro DNA synthesis system in which a synthetic heptaribonucleotide pairs with a unique site on a single-stranded fd DNA molecule and thereby primes the growth of new DNA strands from this single point (Huang, C.-C., and Hearst, J. E. (1980) Anal. Biochem. 103, 127-139). In this report, we use this system to investigate the mechanism by which various bacteriophage T4 DNA replication proteins stimulate the T4 DNA polymerase. We find that with the "polymerase accessory proteins" present (the T4 gene 44/62 and 45 proteins), the DNA polymerase proceeds rather rapidly through the occasional hairpin helices which otherwise interrupt the progress of this enzyme along single-stranded DNA templates. By using a potent inhibitor of the 44/62 ATPase, ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), we have obtained data which suggest that ATP hydrolysis is required for the formation of a polymerase accessory protein-DNA template complex, and that this complex then persists, serving as a sliding clamp which greatly increases the strength of binding between a T4 DNA polymerase molecule and its 3'OH primer template end. The progress of the T4 DNA polymerase though hairpin helices in the DNA template is also stimulated by addition of the T4 helix-destabilizing protein (gene 32 protein). The effect of the 44/62 and 45 proteins is independent of the effect of the 32 protein in this assay, and the rate of polymerase travel over the strongest hairpin helices is increased more than 40-fold in the presence of these four additional proteins.

DNA Replication↗
PubMed1981

Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus.

The product specified by T4 bacteriophage gene 41 is known from genetic analyses to be essential for phage DNA replication in vivo. Correspondingly, the purified gene 41 protein is an essential component of an efficient in vitro DNA replication system reconstructed from seven purified T4 replication proteins; it is required both for the synthesis of short RNA primers (in conjunction with the T4 gene 61 protein) and for the rapid unwinding of the double-helical DNA template at a replication fork. The purified gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity. In this report, we have used this associated GTPase activity as a biochemical probe for the analysis of the interactions between DNA and the 41 protein. Our results suggest that, upon binding GTP, the 41 protein monomer is induced to form a dimer, which can them form a tight complex with single-stranded DNA. Driven by the repeated hydrolysis of GTP molecules, the 41 protein dimer appears to run rapidly along the bound DNA chain. Studies with the synthetic GTP analogue, GTP gamma S, suggest that GTP hydrolysis is required for this 41 protein movement, but that it is not essential for the function of the 41 protein in RNA primer synthesis. In sum, our observations suggest that a 41 protein dimer runs along the lagging strand template at a DNA replication fork; from this position, it functions as a DNA helicase and simultaneously interacts with the T4 gene 61 protein to make the pentaribonucleotide primers which initiate Okazaki pieces at specific primer initiation sites.

DNA Replication↗
PubMed1981

Characterization of RNA primer synthesis in the T4 bacteriophage in vitro DNA replication system.

In the presence of any natural single-stranded DNA, the bacteriophage T4 gene 41 and gene 61 proteins catalyze the synthesis of a group of pentaribonucleotides. These RNA molecules are homogeneous in chain length, but heterogeneous in sequence. In the presence of the other five T4 replication proteins, these pentaribonucleotides are utilized with high efficiency as primers for de novo DNA chain initiations on the lagging strand in the T4 DNA replication process. By using a two-dimensional fractionation system plus a variety of enzymatic analyses, the pentaribonucleotides are found to have either a unique pppApC dinucleotide or a pppG as their starting residue, followed in both cases by many different sequences. However, when natural T4 DNA containing glucosylated 5-hydroxylmethyl cytosine is used as template, only the pppApC-initiated class of RNA primers is made. Alone, the gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity, with nucleoside diphosphate and inorganic phosphate as products. By blocking this GTPase activity with the synthetic GTP analogue, GTP gamma S, we show that limited RNa primer synthesis can occur without this nucleotide hydrolysis by the 41 protein, but that prolonged RNA primer synthesis requires such hydrolysis. This result, viewed in the light of the observations presented in the accompanying paper, suggests that the gene 41 protein acts as a "mobile promoter," perhaps leaving the gene 61 protein to perform the actual polymerization and chain termination functions in the synthetic process.

DNA Replication↗
PubMed1980

Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex.

The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.

Coliphages↗
PubMed1980

Extensive purification of histone acetylase A, the major histone N-acetyl transferase activity detected in mammalian cell nuclei.

A concentrated, DNA-free extract of histone acetylase A was prepared from calf thymus tissues in two simple steps, which exploit the ability of polyethylene glycol to precipitate both nucleic acids and proteins from solutions containing high concentrations of salt (Alberts, B., and Herrick, G. (1971) Methods Enzymol. 21, 198-217). This extract was then chromatographed on four successive columns. The use of 75 microgram/ml of insulin as a carrier protein in all of these later steps, plus the inclusion of 1 M urea in some column buffers, has been useful in improving both the yield and reproducibility of the purification. The highly active enzyme obtained has a molecular weight of about 70,000, and the best fractions could be about 30% pure. Our data indicate that the acetylase A is only a very minor protein in cells, being present in perhaps a few thousand molecules per cell.

Acetyltransferases↗
PubMed1980

Comparative studies of histone acetylation in nucleosomes, nuclei, and intact cells. Evidence for special factors which modify acetylase action.

We have studied the pattern of histone acetylation in intact rat hepatoma tissue culture (HTC) cells, in isolated HTC nuclei, and in chromatin prepared from these cells. The results have been compared with the histone acetylation observed in a reconstituted in vitro system consisting of a variety of purified soluble nucleosomal substrates, [3H]acetyl-CoA, and one of two different purified histone N-acetyltransferases. Acetylase A, a highly purified nuclear enzyme, catalyzed the acetylation of 1) nucleosomally bound histones in the order H4 > H2a = H2b > H3, and 2) free histones in the order H4 > H3 > H2b > H2a. Acetylase B, a cytoplasmic enzyme, modified only free histone H4, and it failed to acetylate histones in nucleosomes. The pattern of histone acetylation obtained by in vitro reaction of purified nucleosomes with the purified nuclear acetylase A differed considerably from the corresponding patterns obtained either by acetate labeling of intact cells, or by the acetyl-CoA labeling of nuclei and crude preparations of nucleosomes, as catalyzed by endogenous chromatin-bound acetylase(s). The most striking difference was in the relative preference for acetylation of histone H4 versus acetylation of histone H3: with the purified acetylase, histone H4 in nucleosomes was acetylated to a much greater extent than was histone H3, whereas the reverse preference was found with the endogenous acetylase(s). This result suggests that either a second nuclear acetylase enzyme, or a separate cofactor for acetylase A, is required for histone H3 acetylation in vivo. In support of this view, we find that the acetylation of histones H4, H2a, and H2b in nuclei is inhibited by urea, salt, or N-ethylmaleimide treatments to a very different extent than is the acetylation of histone H3. By comparing n-butyrate-treated HTC cells with untreated cells, classes of nucleosomes specially accessible and inaccessible to acetylation can be distinguished (Cousens, L. S., Gallwitz, D., and Alberts, B. M. (1979) J. Biol. Chem. 254, 1716-1723). Both types of special nucleosomal reactivities were present in isolated nuclei, but were lost as nucleosomes were purified from these cells. OUr data thus suggest the existence of labile specificity factors or structures, which guide the acetylase(s) to restricted groups of otherwise similar nucleosomes in vivo.

Acetylation↗
PubMed1980

Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex.

More than 50 copies of a phi X174 DNA template can be made in 60 min in an in vitro DNA replication system consisting of seven purfied replication proteins isolated from T4 bacteriophage-infected cells. By transfecting with the DNA products and assaying for the reversion of specific amber mutants, the high degree of base-pairing fidelity in this system is revealed; the in vitro system is also shown to respond to the mutagenic effect of Mn2+ and to display strong base-pair context effects on fidelity, as expected from in vivo studies.

Bacteriophage phi X 174↗
PubMed1980

Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system.

A wide variety of double-stranded DNA templates are replicated extensively in an in vitro DNA replication system containing the purified proteins specified by seven T4 bacteriophage DNA replication genes (32, 41, 43, 44, 62, 45, and 61). In favorable conditions, this multiprotein system catalyzes the synthesis of several copies of the input DNA template in a 30- to 60-min incubation. The replication forks produced in vitro move in a highly processive fashion, at approximately the in vivo rate of 500 nucleotides per s. The DNA synthesized on the lagging side of the in vitro replication fork is made discontinuously, as it is in vivo, giving rise to "Okazaki pieces" averaging some 10,000 nucleotides in length; in contrast, DNA is polymerized in a continuous manner on the leading side of the in vitro fork. Although the mechanism by which the seven-protein in vitro DNA replication system propagates replication forks closely resembles the in vivo mechanism, it lacks the capacity to remove RNA primers, to reseal Okazaki pieces, and to initiate replication forks at defined DNA origins; supplementation of the system with additional T4-specific replication proteins will be required to facilitate these latter three functions.

DNA, Viral↗
PubMed1980

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break.

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.

Animals↗
PubMed1980

Pentaribonucleotides of mixed sequence are synthesized and efficiently prime de novo DNA chain starts in the T4 bacteriophage DNA replication system.

In the presence of single-stranded DNA, the bacteriophage T4 gene 41 and gene 61 proteins catalyze the synthesis of a group of pentaribonucleotides which are homogeneous in chain length but heterogeneous in nucleotide sequence. When single-stranded T4 DNA is used as template, a unique dinucleoside sequence, pppApC, is found at the 5' end of these pentaribonucleotides with the general sequence pppApCpNpNpN. In the presence of the remaining five T4 replication proteins, the pentaribonucleotides can be utilized with high efficiency to prime de novo DNA chain starts; as a result, the vast majority of them can be detected at the 5' end of newly made DNA molecules in an unaltered form. There are multiple, but specific, sites at which new DNA chains are primed in this way on a natural single-stranded DNA. Because identical RNA primers have been isolated from the 5' end of the Okazaki fragments made in T4-infected cells, we suggest that the T4 gene 41 and gene 61 proteins also make the pentaribonucleotides that prime de novo T4 DNA chain starts in vivo during lagging strand DNA synthesis.

Base Sequence↗
PubMed1979

The use of DNA-cellulose for analyzing histone-DNA interactions. Discovery of nucleosome-like histone binding to single-stranded DNA.

In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones.

Animals↗