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Biomedical subjects

J W Chase

Publications and source records attributed to J W Chase.

At least 37 records · Page 2Linked to original sources
PubMed1987

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides.

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.

DNA-Binding Proteins↗
PubMed1987

Triplet state sublevel kinetics of tryptophan 54 in the complex of Escherichia coli single-stranded DNA binding protein with single-stranded poly(deoxythymidylic) acid.

The individual sublevel kinetics of the lowest triplet state of tryptophan 54 (Trp 54) which is highly perturbed in the complex of Escherichia coli single-stranded DNA binding protein (Eco SSB) with poly(deoxythymidylic) acid (poly[dT]) have been studied by optically detected magnetic resonance (ODMR) spectroscopy. The triplet sublevel decay constants of Trp 54, kx, ky, kz, are 0.99, 0.072, and 0.045 s-1, respectively, in the poly(dT) complex of a point-mutated Eco SSB in which Trp 88 is substituted by phenylalanine. Tx is the only radiative triplet sublevel. Negative polarity of the Tx----Tz and Tx----Ty phosphorescence-detected ODMR signals results from the steady state population pattern, nx greater than ny, nz, and implies that the relations, px greater than or equal to 14py, and px greater than or equal to 22pz exist for the relative populating rates. Spin-orbit coupling between radiative singlet states and the Tx sublevel of the lowest triplet state of Trp 54 is enhanced selectively upon complexing of Eco SSB with poly(dT).

DNA-Binding Proteins↗
PubMed1987

Effects of various single-stranded-DNA-binding proteins on reactions promoted by RecA protein.

To relate the roles of Escherichia coli SSB in recombination in vivo and in vitro, we have studied the mutant proteins SSB-1 and SSB-113, the variant SSBc produced by chymotryptic cleavage, the partially homologous variant F SSB (encoded by the E. coli sex factor), and the protein encoded by gene 32 of bacteriophage T4. All of these, with the exception of SSB-1, augmented both the initial rate of homologous pairing and strand exchange promoted by RecA protein. From these and related observations, we conclude that SSB stimulates the initial formation of joint molecules by nonspecifically promoting the binding of RecA protein to single-stranded DNA; that SSB plays no role in synapsis of the RecA nucleoprotein filament with duplex DNA; that stimulation of strand exchange by SSB is similarly nonspecific; and that all members of the class of proteins represented by SSB, F SSB, and gene 32 protein may play equivalent roles in making single-stranded DNA more accessible to RecA protein.

DNA, Single-Stranded↗
PubMed1986

Escherichia coli exonuclease VII. Cloning and sequencing of the gene encoding the large subunit (xseA).

We have determined the sequence of the gene encoding the large subunit of Escherichia coli exonuclease VII (xseA) and the amino acid sequence of the protein it encodes. The coding region of the xseA gene is 1368 base pairs. The protein encoded by the gene contains 456 amino acids and has a calculated molecular weight of 51,823. The promoter for xseA is close to that for guaB, and these two genes are transcribed in opposite directions: xseA clockwise and guaB counterclockwise on the standard E. coli genetic map. The cloned xseA gene can complement an xseA deletion mutant strain. In an xseA+ genetic background production of large quantities of the xseA gene product appeared to decrease the amount of exonuclease VII activity in cell extracts. In fact, no exonuclease VII activity at all could be detected following induction of strains in which the xseA gene was under lambda pL regulation. These observations suggest that the proper ratio of the large and small exonuclease VII subunits must be maintained in order to produce active enzyme.

Amino Acid Sequence↗
PubMed1986

DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization.

A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.

Adenosine Triphosphate↗
PubMed1986

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis.

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.

Amino Acid Sequence↗
PubMed1986

Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli.

Bacteriophage T4 gene 32 encodes a single-stranded DNA binding protein required for T4 DNA replication, recombination, and repair. Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability. In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P. A repetitive A + T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation. To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region. The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis. In vectors containing the lambda leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being depressed at g32P concentrations that repress the wild-type gene 32 transcripts.

Bacteriophage lambda↗
PubMed1985

Intermediates in homologous pairing promoted by recA protein. Isolation and characterization of active presynaptic complexes.

recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA. This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized. At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA. Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein. Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.

Adenosine Diphosphate↗
PubMed1985

A monoclonal antibody that recognizes the functional domain of Escherichia coli single-stranded DNA binding protein that includes the ssb-113 mutation.

We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site. Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein. Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein. In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability. Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB. This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113. The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.

Antibodies, Monoclonal↗
PubMed1984

Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene. Expression of the ssb-1 gene under lambda pL regulation.

The ssb-1 gene encoding a mutant single-stranded DNA binding protein (SSB-1) has been cloned into a vector placing its expression under lambda pL regulation. This construction results in more than 100-fold increased expression of the mutant protein following temperature induction. Tryptic peptide analysis of the mutant protein by high-pressure liquid chromatography and solid-phase protein sequencing has shown that the ssb-1 mutation results in these substitution of tyrosine for histidine at residue 55 of SSB. This change could only occur in one step by a C----T transition in the DNA sequence which has been confirmed. Physicochemical studies of the homogeneous mutant protein have shown that in contrast to that of the wild-type SSB, the tetrameric structure of SSB-1 is unstable and gradually dissociates to monomer as the protein concentration is decreased from about 10 microM to less than 0.5 microM. The SSB-1 tetramer appears to be stable to elevated temperature (45 degrees C) but the monomer is not. We estimate the normal cellular concentration of SSB-1 (single chromosomal gene) to be 0.5-1 microM. Thus, there is a plausible physical explanation for our previous finding that increased expression of ssb-1 reverses the effects of a single gene (chromosomal) copy amount of SSB-1 (Chase, J.W., Murphy, J.B., Whittier, R.F., Lorensen, E., and Sninsky, J.J. (1983) J. Mol. Biol. 164, 193-211). However, even though the in vivo effects of ssb-1 and most of the in vitro defects of SSB-1 protein are reversed simply by increasing SSB-1 protein concentration, the mutant protein is not as effective a helix-destabilizing protein as wild-type SSB as measured by its ability to lower the thermal melting transition of poly[d-(A-T)].

Chromatography, Affinity↗
PubMed1984

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking.

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.

Amino Acids↗
PubMed1984

Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein. Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies.

The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene. Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB. This change could only occur in one step by a C leads to T transition in the DNA sequence. Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly[d(A-T)]. In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J. W., Murphy, J. B., Whittier, R. F., Lorensen, E., and Sninsky, J. J. (1983) J. Mol. Biol. 164, 193-211). Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K. R., Chase, J. W., and Rupp, W. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4274-4278). The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC.

Amino Acid Sequence↗
PubMed1984

Isolation of uvrA mutation on a multicopy plasmid: preliminary characterization of the mutant protein.

A new uvrA mutation (uvrA276) has been isolated on a multicopy plasmid and shown to reside within the region of the uvrA gene defined by the KpnI to SalI endonuclease sites. The protein produced by the uvrA276 mutant gene is identical in size to the wild-type protein and binds to single-stranded DNA under the same conditions as the wild-type protein. However, extracts prepared from strains containing this mutant are deficient at incision of DNA that has been irradiated with UV light.

Bacterial Proteins↗
PubMed1983

Limited proteolysis studies on the Escherichia coli single-stranded DNA binding protein. Evidence for a functionally homologous domain in both the Escherichia coli and T4 DNA binding proteins.

Limited proteolysis can be used to remove either 42 or 62 amino acids at the COOH terminus of the 18,873-dalton Escherichia coli single-stranded DNA binding protein (SSB). Since poly(dT), but not d(pT)16, increases the rate of this reaction, it appears that cooperative SSB binding to single-stranded DNA (ssDNA) is associated with a conformational change that increases the exposure of the COOH terminus to proteolysis. As a result of this DNA-induced conformational change, we presume that the COOH-terminal region of SSB will become more accessible for interacting with other proteins that utilize the SSB:ssDNA complex as a substrate and that are involved in E. coli DNA replication, repair, and recombination. Removal of this COOH-terminal domain from SSB results in a stronger helix-destabilizing protein which suggests this region may be important for controlling the ability of SSB to denature double-stranded DNA. Since similar results have previously been reported for the bacteriophage T4 gene 32 protein (Williams, K.R., and Konigsberg, W. (1978) J. Biol. Chem. 253, 2463-2470; Hosoda, J., and Moise, H. (1978) J. Biol. Chem. 253, 7547-7555), the acidic, COOH-terminal domains of these two single-stranded DNA binding proteins may be functionally homologous. Preliminary evidence is cited that suggests other prokaryotic and eukaryotic DNA binding proteins may contain similar functional domains essential for controlling their ability to invade double helical DNA.

Amino Acid Sequence↗
PubMed1983

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli.

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene; the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage lambda +, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA- mutant strains and will provide a means of transferring ssb- mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.

Bacterial Proteins↗
PubMed1983

DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein.

Mutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair. To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb+, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions. As is true in ssb+ strains tif-1 (recA441) was found to allow thermal induction of prophage lambda + and Weigle reactivation in ssb-1 and ssb-113 strains. Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage lambda + at 30 degrees C. Strains carrying the recAo281 allele were also constructed. This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA- alleles without restoring SOS (error-prone) repair. In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb- mutation. In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain. The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation. These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.

Bacterial Proteins↗