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N G Nossal

Publications and source records attributed to N G Nossal.

At least 37 records · Page 2Linked to original sources
PubMed1991

Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.

T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.

Adenosine Triphosphate↗
PubMed1991

Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro.

RNase H activity increases markedly after bacteriophage T4 infection of Escherichia coli MIC2003, an RNase H-deficient host. We have extensively purified the RNase H from these T4-infected cells and have shown that the RNase H activity copurifies with a 5' to 3' DNA exonuclease activity. The N-terminal sequence of a 35-kDa protein copurifying with the RNase H activity matches the terminus of the predicted product of an open reading frame (designated ORF A or 33.2) upstream of T4 gene 33, identified previously by Hahn and co-workers (Hahn, S., Kruse, U., and Rüger, W. (1986) Nucleic Acids Res. 14, 9311-9327). Plasmids containing ORF A under the control of the T7 promoter express RNase H and 5' to 3' DNA exonuclease activities as well as a protein that comigrates on sodium dodecyl sulfate-polyacrylamide gels with the 35-kDa protein present in the RNase H purified from T4-infected cells. T4 RNase H removes the pentamer RNA primers from DNA chains initiated by the T4 primase-helicase (gene products 61 and 41). Addition of T4 RNase H and T4 DNA ligase leads to extensive joining of discontinuous lagging strand fragments in the T4 DNA replication system in vitro.

Amino Acid Sequence↗
PubMed1989

Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro.

The bacteriophage T4 gene 41 protein is a 5' to 3' DNA helicase which unwinds DNA ahead of the growing replication fork and, together with the T4 gene 61 protein, also functions as a primase to initiate DNA synthesis on the lagging strand. Proteolytic cleavage by trypsin approximately 20 amino acids from the COOH terminus of the 41 protein produces 41T, a 51,500-dalton fragment (possibly still associated with small COOH-terminal fragments) which still retains the ssDNA-stimulated GTPase (ATPase) activity, the 61 protein-stimulated DNA helicase activity, and the ability to act with 61 protein to synthesize pentaribonucleotide primers. In the absence of the T4 gene 32 ssDNA binding protein, the primase-helicase composed of the tryptic fragment (41T) and 61 proteins efficiently primes DNA synthesis on circular ssDNA templates by the T4 DNA polymerase and the three T4 polymerase accessory proteins. In contrast, the 41T protein is defective as a helicase or a primase component on 32 protein-covered DNA. Thus, unlike the intact protein, 41T does not support RNA-dependent DNA synthesis on 32 protein-covered ssDNA and does not stimulate strand displacement DNA synthesis on a nicked duplex DNA template. High concentrations of 32 protein strongly inhibit RNA primer synthesis with either 41 T or intact 41 protein. The 44/62 and 45 polymerase accessory proteins (and even the 44/62 proteins to some extent) substantially reverse the 32 protein inhibition of RNA primer synthesis with intact 41 protein but not with 41T protein. We propose that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T4 replication complex. When this region is altered, as in 41T protein, the protein is unable to assemble a functional primase-helicase in the replication complex. An easy and rapid purification of T4 41 protein produced by a plasmid encoding this gene (Hinton, D. M., Silver, L. L., and Nossal, N. G. (1985) J. Biol. Chem. 260, 12851-12857) is also described.

Bacteriophage phi X 174↗
PubMed1989

Characterization of the bacteriophage T4 gene 41 DNA helicase.

The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro. We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L. L., and Nossal, N. G. (1982) J. biol. Chem. 257, 12426-12434). The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein. We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork. Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template). This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork. As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished. However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs). These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein. A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity). In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.

Adenosine Triphosphate↗
PubMed1987

Bacteriophage T4 DNA primase-helicase. Characterization of oligomer synthesis by T4 61 protein alone and in conjunction with T4 41 protein.

The bacteriophage T4 41 and 61 proteins function as a primase-helicase which in vitro both unwinds double-stranded DNA and synthesizes the pentaribonucleotides used to initiate DNA synthesis on the lagging strand. We demonstrate that 61 protein alone possesses a weak DNA template-dependent oligomer synthesizing activity, whose products differ in size and nucleotide specificity from those made by the 61 and 41 proteins together. We have previously shown that the 61 and 41 proteins make primarily ribonucleotide pentamers of the sequence pppApC(pN)3, although some pentamers beginning with G were also detected on phi X174 single-stranded DNA. The pentamers pppApC(pN)3 have also been shown to initiate T4 DNA chains in vivo (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861). We now show that in contrast, the major products made by 61 protein alone on phi X174 DNA with [alpha-32P]CTP and the other three ribonucleoside triphosphates are not pentamers, but the dimers pppApC and pppGpC. In addition, minor amounts of products from 3 to approximately 45 nucleotides in length are also synthesized. Unlike the 61/41 protein reaction, 61 protein alone can substitute dATP or dGTP for ATP or GTP. Addition of 41 protein greatly stimulates oligomer synthesis, especially the synthesis of products made with ATP and CTP and products 5 nucleotides in length. Thus, both 61 and 41 proteins are needed to obtain efficient synthesis of the biologically relevant pentamers pppApC(pN)3. We demonstrate that the glucosylated hydroxymethylcytosines present in T4 DNA do not support the initiation of primer synthesis by the 61 protein on this template. With glycosylated hydroxymethyl T4 DNA, pppApC but not pppGpC oligomers are detected. If the T4 DNA is modified by hydroxymethylation but not glucosylation, pppApC and only a trace of pppGpC products are seen. In the accompanying paper (Nossal, N.G., and Hinton, D.M. (1987) J. Biol. Chem. 262, 10879-10885), we examine DNA synthesis primed by 61 protein in the absence of 41 protein.

DNA Helicases↗
PubMed1987

Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein.

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.

Bacteriophage phi X 174↗
PubMed1986

Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein.

The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication. We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein. This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease. The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA. These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein. The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene.

Adenosine Triphosphatases↗
PubMed1985

Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein.

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein.

Ammonium Sulfate↗
PubMed1985

Bacteriophage T4 DNA replication protein 61. Cloning of the gene and purification of the expressed protein.

In vitro, a bacteriophage T4 primase composed of T4 61 and 41 proteins, catalyzes the formation of pentaribonucleotides used to initiate DNA synthesis on single-stranded DNA. We have determined that cells containing a plasmid with the T4 DNA from 18.68 to 15.05 map units express an activity that substitutes for authentic 61 protein in vitro in catalyzing primer-dependent DNA synthesis with six other T4 DNA replication proteins. This result establishes that this region, genetically assigned to gene 61, is the structural gene for the priming protein. Cells containing a plasmid with gene 61 downstream of the strong phage lambda promoter PL and the antitermination site nutL produce 100-fold more 61 protein than T4-infected cells. We have developed an improved purification procedure which yields 100 mg of homogeneous, active protein from 178 g of these cells. In the plasmid, the T4 DNA downstream of gene 61 expresses a protein of 30,000 daltons. This protein may be the T4 DNA adenine methylase (dam) gene product, since Schlagman and Hattman (Schlagman, S. L. and Hattman, S. (1983) Gene 22, 139-156) have shown that this activity is expressed by plasmids containing T4 DNA from this region. In the PL, nutL vector, the expression of both the 30,000-dalton and 61 proteins is enhanced up to 20-fold by the presence of the phage lambda N protein, a transcription antitermination protein, suggesting that expression of the T4 DNA in the plasmid may be regulated transcriptionally. In addition, in both N+ and N- cells, the level of 61 protein, whose gene is proximal to PL on the plasmid, is lower than that of the product of the promoter distal 30,000-dalton protein gene. This result suggests that, at least in the plasmid construction, the expression of 61 protein may also be regulated after transcription.

Bacteriophage lambda↗
PubMed1984

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product.

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.

Amino Acid Sequence↗
PubMed1982

Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase.

Bacteriophage T4 gene 41 protein is one of the two phage proteins previously shown to be required for the synthesis of the pentaribonucleotide primers which initiate the synthesis of new chains in the T4 DNA replication system. We now show that a DNA helicase activity which can unwind short fragments annealed to complementary single-stranded DNA copurifies with the gene 41 priming protein. T4 gene 41 is essential for both the priming and helicase activities, since both are absent after infection by T4 phage with an amber mutation in gene 41. A complete gene 41 product is also required for two other activities previously found in purified preparations of the priming activity: a single-stranded DNA-dependent GTPase (ATPase) and an activity which stimulates strand displacement synthesis catalyzed by T4 DNA polymerase, the T4 gene 44/62 and 45 polymerase accessory proteins, and the T4 gene 32 helix-destabilizing protein (five-protein reaction). The 41 protein helicase requires a single-stranded DNA region adjoining the duplex region and begins unwinding at the 3' terminus of the fragment. There is a sigmoidal dependence on both nucleotide (rGTP, rATP) and protein concentration for this reaction. 41 Protein helicase activity is stimulated by our purest preparation of the T4 gene 61 priming protein, and by the T4 gene 44/62 and 45 polymerase accessory proteins. The direction of unwinding is consistent with the idea that 41 protein facilitates DNA synthesis on duplex templates by destabilizing the helix as it moves 5' to 3' on the displaced strand.

Adenosine Triphosphate↗
PubMed1982

Purification of bacteriophage T4 gene 61 protein. A protein essential for synthesis of RNA primers in the T4 in vitro DNA replication system.

An activity dependent on an intact bacteriophage T4 gene 61 is required, along with the T4 gene 41 protein, for the synthesis of ribonucleotide primers in an in vitro T4 DNA replication system. In this paper, we present a method for purification of the protein catalyzing this gene 61-dependent activity based on an assay for primer-dependent DNA synthesis by the T4 DNA replication proteins. The T4 gene 32 helix-destabilizing protein influences the chromatographic behavior of 61 protein. The purification of 61 protein to near homogeneity by the scheme presented requires the presence of 32 protein in crude extracts. The isolated 61 protein is basic, with a molecular weight of 44,000, and is active as a monomer. Ribonucleotide primer synthesis shows a linear dependence on 61 protein concentration, but a sigmoidal dependence on 41 protein concentration. The dependence on 41 protein concentration is linear, however, if the 41 protein is first "activated" by incubation at high concentration in the presence of rGTP. Using this "activated" 41 protein and purified 61 protein, we show a stoichiometric relationship between the two proteins in the priming reaction consistent with the existence of a priming complex comprising an oligomer of 41 protein and a 61 protein monomer.

Chromatography, Affinity↗
PubMed1982

Bacteriophage T4 gene 45. Sequences of the structural gene and its protein product.

Bacteriophage T4 gene 45 codes for a protein whose functions are required for both T4 DNA replication and T4 late gene transcription. To facilitate studies of the interactions of 45 protein with the T4 DNA replication complex and with RNA polymerase, we have determined the primary structure of 45 protein. The amino acid sequence of 45 protein has been determined by correlating nucleotide sequence analysis of gene 45 with protein chemistry studies of 45 protein. Our studies indicate that gene 45 codes for a polypeptide containing 227 amino acids, with a calculated Mr = 24,710. The coding region of gene 46 is preceded by a putative promoter containing sequences which are homologous to Escherichia coli RNA polymerase recognition and binding regions. In addition, there are sequence similarities in the translation initiation regions of gene 45 and the rIIB gene, which may relate to their common regulation by regA protein.

Base Sequence↗
PubMed1980

RNA priming of DNA replication by bacteriophage T4 proteins.

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.

Base Sequence↗
PubMed1979

DNA replication with bacteriophage T4 proteins. Purification of the proteins encoded by T4 genes 41, 45, 44, and 62 using a complementation assay.

The proteins encoded by bacteriophage T4 genes 41, 45, 44, and 62 are known from the genetic studies of Epstein et al. ((1963) Cold Spring Harbor Symp. Quant. Biol. 28, 375--394) to be required for viral DNA synthesis. A convenient assay for each of these proteins is described which is based on the specific stimulation by each protein of DNA synthesis in extracts of Escherichia coli infected with mutants of bacteriophage T4 unable to make that protein. The T4 41 protein, 45 protein, and the complex of the 44 and 62 proteins have been highly purified. For each protein there is co-chromatography during the final purification step of (i) activity in the complementation assay, (ii) activity required for DNA synthesis with other purified T4 proteins, and (iii) a subunit of the size previously identified as that of the corresponding gene product.

Coliphages↗