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Biomedical subjects

E G Frank

Publications and source records attributed to E G Frank.

30 records · Page 2Linked to original sources
PubMed1996

Arrested DNA replication in Xenopus and release by Escherichia coli mutagenesis proteins.

Xenopus oocytes and oocyte nuclear extracts repair ultraviolet photoproducts on double-stranded (ds) DNA and replicate single-stranded (ss) to ds DNA. M13 ss DNA molecules containing cyclobutane pyrimidine dimers were maintained but not replicated in Xenopus oocytes yet were replicated in progesterone-matured oocytes. The replication arrest functioned only in cis. The replication arrest was alleviated by injection into oocytes of messenger RNAs encoding the prokaryotic mutagenesis proteins UmuD'C or MucA'B. These results may help explain how cells stabilize repair or replication events on DNA with unrepairable lesions.

Animals↗
PubMed1996

Structure of the UmuD' protein and its regulation in response to DNA damage.

For life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information. In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response'. For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication. Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD' and UmuC (umu: UV mutagenesis). Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD'. To understand this system in more detail, we have determined the crystal structure of the E. coli UmuD' mutagenesis protein at 2.5 A resolution. Globular heads folded in an unusual Beta-structure associate to form molecular dimers, and extended amino-terminal tails associate to produce crystallized filaments. The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response.

Amino Acid Sequence↗
PubMed1996

Production and crystallization of a selenomethionyl variant of UmuD', an Escherichia coli SOS response protein.

Crystals of both native and mutant Escherichia coli UmuD' protein were obtained using the hanging drop method. Soaking the native crystals in solutions of heavy metal ions failed to produce good isomorphous derivatives, and selenomethionine substituted wild-type protein did not crystallize under conditions that gave native crystals. Site-directed mutagenesis was used to change the penultimate residue, a methionine amino acid, to either a valine or a threonine amino acid. Crystals were subsequently obtained from these mutant proteins with and without selenomethionine incorporation. Crystals of the native, the mutant, and the selenomethionine incorporated protein were all similar, crystallizing in the P4(1)2(1)2 space group.

Amino Acid Sequence↗
PubMed1996

In vivo stability of the Umu mutagenesis proteins: a major role for RecA.

The Escherichia coli Umu proteins play critical roles in damage-inducible SOS mutagenesis. To avoid any gratuitous mutagenesis, the activity of the Umu proteins is normally kept to a minimum by tight transcriptional and posttranslational regulation. We have, however, previously observed that compared with an isogenic recA+ strain, the steady-state levels of the Umu proteins are elevated in a recA730 background (R. Woodgate and D. G. Ennis, Mol. Gen. Genet. 229:10-16, 1991). We have investigated this phenomenon further and find that another coprotease-constitutive (recA*) mutant, a recA432 strain, exhibits a similar phenotype. Analysis revealed that the increased steady-state levels of the Umu proteins in the recA* strains do indeed reflect an in vivo stabilization of the proteins. We have investigated the basis for the phenomenon and find that the mutant RecA* protein stabilizes the Umu proteins by not only converting the labile UmuD protein to the much more stable (and mutagenically active) UmuD' protein but by directly stabilizing UmuD' itself. In contrast, UmuC does not appear to be directly stabilized by RecA* but is instead dramatically stabilized in the presence of UmuD'. On the basis of these observations, we suggest that formation of a UmuD'C-RecA*-DNA quaternary complex protects the UmuD'C proteins from proteolytic degradation and as a consequence helps to promote the switch from error-free to error-prone mechanisms of DNA repair.

Bacterial Proteins↗
PubMed1994

Isolation and characterization of novel plasmid-encoded umuC mutants.

Most inducible mutagenesis in Escherichia coli is dependent upon the activity of the UmuDC proteins. The role of UmuC in this process is poorly understood, possibly because of the limited number of genetically characterized umuC mutants. To better understand the function of the UmuC protein in mutagenic DNA repair, we have isolated several novel plasmid-encoded umuC mutants. A multicopy plasmid that expressed UmuC at physiological levels was constructed and randomly mutagenized in vitro by exposure to hydroxylamine. Mutated plasmids were introduced into the umu tester strain RW126, and 16 plasmids that were unable to promote umuC-dependent spontaneous mutator activity were identified by a colorimetric papillation assay. Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremely useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

Bacterial Proteins↗
PubMed1993

Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein.

In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis. This role can be separated genetically into three steps: (i) depression of the SOS regulon by mediating the posttranslational cleavage of the LexA repressor, (ii) activation of UmuD'-like proteins by mediating cleavage of the UmuD-like proteins, and (iii) a direct step, possibly to interact with and to target the Umu-like mutagenesis proteins to lesions in DNA. We have analyzed RecA's third role biochemically using protein affinity chromatography and an agarose-based DNA mobility-shift assay. RecA730 protein from a crude cell extract was specifically retained on UmuD and UmuD' protein affinity columns, suggesting that these proteins physically interact. Normally, neither UmuD nor UmuD' shows any affinity for DNA. In the presence of RecA protein, however, UmuD and UmuD' were targeted to DNA. RecA1730 protein, which is defective for UmuD' but proficient for MucA'-promoted mutagenesis, showed a dramatically reduced capacity to target UmuD' to DNA but was able to target a significant portion of MucA' to DNA. These data support the suggestion that the direct role of RecA protein in SOS-induced mutagenesis is to interact with and target the Umu-like mutagenesis proteins to DNA.

Bacterial Proteins↗
PubMed1991

The number of cooperative regions (energetic domains) in a pepsin molecule depends on the pH of the medium.

The technique of scanning microcalorimetry was used to study the effect exerted by ethanol and by the pH of the medium on the number and size of cooperative regions in a pepsin molecule. Ethanol addition lowered the temperature of protein denaturation, but did not change the number of energetic domains. The number of thermodynamic cooperative units (determined as a delta Hcal to delta Heff ratio) was reduced from four to two when the pH changed from 6.7 to 2.0. As was demonstrated using the CD technique, this process involved no changes either in the secondary structure or in the local surroundings of aromatic amino acids. Therefore, variations in the cooperative properties of a pepsin globule at different pH values are associated with the electrostatic interactions of individual parts of the molecule.

Animals↗
PubMedDate not supplied

[The existence of the ribonucleoprotein branching enzyme in rabbit skeletal muscle and ribozyme corresponding to it].

Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously.

1,4-alpha-Glucan Branching Enzyme↗
PubMedDate not supplied

[Number of cooperative regions (energy domains) in the pepsin molecule depends on the pH of the medium].

Ethanol and pH influence on the number and dimensions of cooperative regions in pepsin molecule was studied by scanning microcalorimetry. It is shown that ethanol solution causes a decrease of temperature of protein denaturation but does not influence the number of energetic domains. While changing pH from 6.7 to 2.0 the number of thermodynamic cooperative units (defined as the ratio delta Hcal/delta Heff) decreases from four to two. This process, as shown by CD technique, is followed by changes neither in the secondary structure, nor in the local environment of aromatic amino acids. A conclusion is made that the distinctions in cooperative characteristics of the protein globule at different pH are determined by electrostatic interactions of separate parts of the molecule.

Animals↗
PubMedDate not supplied

[Determination of the heat expansion coefficient of pepsin crystals. Anisotropy of conformation dynamics of the enzyme globule].

The microscopic study of heat expansion of monoclinic pepsin crystals was performed in temperature interval of -120 degrees C to +20 degrees C. The coefficient of heat expansion along the C axis was shown to be 8-10 times as large as along the B axis. This dynamics anisotropy is interpreted to reflect the domain mobility of the molecule.

Chemical Phenomena↗
PubMedDate not supplied

[A comparative study of the thermal stability of phenylalanyl tRNA synthetase from Escherichia coli MRE-600 and Thermus thermophilus HB 8].

The thermal stability of phenylalanyl-tRNA-synthetase (PTS) from E. coli and T.thermophilus HB 8 was studied in solution at various conditions by scanning microcalorimetry. It has been shown that the value of heating rate, concentration of the enzyme and Mg2+ ions in the solution affects the parameters of thermal denaturation of both enzymes. The higher thermal stability of PTS from T. thermophilus was observed as well as the independence of its properties upon broad variations of experimental conditions. The role of thermostability of the enzymes are discussed with respect to the biological properties of E. coli and T.thermophilus.

Calorimetry, Differential Scanning↗