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Biomedical subjects

M O'Donnell

Publications and source records attributed to M O'Donnell.

At least 19 recordsLinked to original sources
PubMed1992

Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli generates the induced, multigene SOS response. One component of the SOS response is a marked increase in mutation rate, dependent on RecA protein and the induced mutagenesis proteins UmuC and UmuD. A variety of previous indirect approaches have indicated that SOS mutagenesis results from replicative bypass of the DNA lesion by DNA polymerase III (pol III) holoenzyme in a reaction mediated by RecA, UmuC, and a processed form of UmuD termed UmuD'. To study the biochemistry of SOS mutagenesis, we have reconstituted replicative bypass with a defined in vitro system containing purified protein and a DNA substrate with a single abasic DNA lesion. The replicative bypass reaction requires pol III, UmuC, UmuD', and RecA. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering UmuD' in a heterodimer. Our experiments demonstrate directly that the UmuC-UmuD' complex and RecA act to rescue an otherwise stalled pol III holoenzyme at a replication-blocking DNA lesion.

Bacterial Proteins↗
PubMed1992

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.

Autoradiography↗
PubMed1992

Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp.

The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta [and epsilon] processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.

Amino Acid Sequence↗
PubMed1992

Accessory protein function in the DNA polymerase III holoenzyme from E. coli.

DNA polymerases which duplicate cellular chromosomes are multiprotein complexes. The individual functions of the many proteins required to duplicate a chromosome are not fully understood. The multiprotein complex which duplicates the Escherichia coli chromosome, DNA polymerase III holoenzyme (holoenzyme), contains a DNA polymerase subunit and nine accessory proteins. This report summarizes our current understanding of the individual functions of the accessory proteins within the holoenzyme, lending insight into why a chromosomal replicase needs such a complex structure.

DNA Polymerase III↗
PubMed1992

The horn flap: a curved V-Y advancement flap with lateral pedicle.

The Horn Flap, a curved V-Y advancement flap with a radially based subcutaneous pedicle, has proven to be a versatile, reliable method of reconstruction following excision of small facial lesions, particularly in the medial canthal area. Entry of the pedicle on the radial, or concave, side makes significant advancement possible.

Dermatologic Surgical Procedures↗
PubMed1992

Lipoprotein Lp(a) levels are reduced by danazol, an anabolic steroid.

Serum levels of lipids, lipoproteins and apolipoproteins were measured in 26 premenopausal women with endometriosis both before and after six months therapy with the anabolic steroid danazol (600 mg/day) and in 15 untreated women who acted as controls. No changes were seen in the control group over six months. In women treated with danazol, mean levels of low density lipoprotein (LDL) cholesterol increased by 36% while those of high density lipoprotein (HDL) cholesterol decreased by 46%, changes characteristic of androgenic steroids. In contrast to this potentially detrimental lipoprotein profile, lipoprotein(a) [Lp(a)] levels were reduced by 78.6% +/- 24.0% (mean +/- S.D.) in women taking danazol. These dramatic changes in Lp(a) levels correlated with baseline Lp(a) levels but not with changes in LDL or HDL. Anabolic steroids such as danazol appear to be powerful modulators of serum Lp(a) concentrations. This could be due to direct effects on Lp(a) metabolism, or secondary to the effects of these steroids on insulin metabolism or on the coagulation and fibrinolysis system.

Adolescent↗
PubMed1992

HLA phenotype is a factor in determining rate of disease progression and outcome in HIV-1-infected individuals.

HLA allele frequencies were examined for possible association(s) with the rate of disease progression and with the disease outcome (AIDS diagnosis) in a population of HIV-1-infected individuals. Certain alleles were associated with the relative rate of CD4+ T-cell decline. Association of particular alleles with several disease outcomes associated with infection was also observed. It is important to keep these two aspects (disease progression, AIDS diagnosis) separate when studying HLA in the HIV-1-infected population. Alleles that may play a role in the rate of virus speed by effecting the immune response may be different from those found to be associated with a particular disease. We feel that the only truly informative data, in this regard, can be generated from a relative precise determination of the time of infection (to study disease progression) and adequate numbers of individuals with specific diseases to study specific disease association. If such data can be generated we will have a much better understanding of the pathogenetic process(es) of HIV-1 infection.

Alleles↗
PubMed1992

Lipid and lipoprotein (a) concentrations in renal transplant patients.

Lipid and lipoprotein concentrations, including lipoprotein (a), were measured in 67 clinically stable renal allograft recipients and compared with age- and sex-matched controls. Median lipoprotein (a) concentrations were significantly elevated in the transplant group (P = 0.048), with the distribution of apoprotein (a) isoforms being similar between the two groups. The transplant group also demonstrated significant elevations in cholesterol (P less than 0.0001), triglycerides (P = 0.0007) and low-density lipoprotein cholesterol (P less than 0.0001). There was no significant difference in high-density lipoprotein cholesterol concentrations between the groups although there was the expected tendency for higher values in females. Lipoprotein abnormalities are common following renal transplantation and these patients also demonstrate elevated lipoprotein (a) values. This unique lipoprotein is known to be atherogenic and may contribute to the development of vascular disease, which is a common mode of death in these patients.

Adolescent↗
PubMed1992

EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed.

Antigens, Viral↗
PubMed1992

A new technique examines the connective tissue framework in normal testis and in testis cancer.

A method of cellular digestion known as Etching facilitated scanning electron microscopy (SEM) of the connective tissue framework in normal testis and in cases of testis cancer. The collagen in the seminiferous tubules in normal testis has distinct lamellae and a well-defined outer border. In the germ cell tumours examined, the tubular connective tissue appeared hypertrophied, the distinct outer border was maintained in a seminoma specimen unlike in a case of non-seminoma. In a specimen of non-germ cell lymphoma there was gross distortion of the tubular connective tissue. These apparent variations in the collagen framework may have implications regarding local tumour development.

Collagen↗
PubMed1992

Synthetic aperture imaging using a Lagrange based filtering technique.

Synthetic aperture imaging using a catheter based, circular phased array providing high resolution, dynamic focusing has been explored. Due to the high input impedance and low signal-to-noise ratio (SNR) of a classic single element synthetic aperture system, multi-element synthetic aperture processing has been proposed with SNR improvement of about 8 dB for a 33 element aperture. Reconstruction in this case uses an optimal filtering approach based on minimizing the mean square error between filter output and desired beam pattern. This approach, however, does not directly control both mainlobe beamwidth and sidelobe levels. To overcome this problem, a Lagrange based filter design technique has been developed that not only satisfies the minimum energy criterion, but also constrains sidelobe levels under a certain threshold. The new technique provides better spatial and contrast resolution. Both the mathematical formulation and simulation results are presented.

Algorithms↗
PubMed1991

Epstein-Barr nuclear antigen 1 mediates a DNA loop within the latent replication origin of Epstein-Barr virus.

Epstein-Barr virus-encoded nuclear antigen 1 (EBNA-1) binds and activates the viral latent origin of DNA replication, oriP. We have used electron microscopy to examine the assembly of EBNA-1 onto oriP. The oriP region consists of two essential elements separated by approximately 1 kilobase pair of DNA. One element contains 20 tandom EBNA-1 binding sites [called the family of repeats (FR)] and serves to activate initiation of replication at the dyad symmetry (DS) element, which contains 4 EBNA-1 binding sites. Titration of homogeneous EBNA-1 produced in baculovirus (bEBNA-1) onto oriP DNA showed an order to the assembly of bEBNA-1 onto oriP. At low concentrations, bEBNA-1 was located exclusively on the FR element. As the level of bEBNA-1 was raised, a loop between the FR and DS elements became the most prevalent DNA-protein complex. These data suggest protein-mediated DNA looping may play a role in activating latent-phase replication of the Epstein-Barr virus.

Antigens, Viral↗
PubMed1991

Treatment of adult acute lymphoblastic leukemia with intensive cyclical chemotherapy: a follow-up report.

We treated 109 patients with adult acute lymphoblastic leukemia (ALL) diagnosed by histochemical and immunologic techniques. Patients were excluded only for age greater than 50 years and Burkitt's leukemia. Treatment included a four-drug remission induction phase followed by alternating cycles of noncrossresistant chemotherapy and prolonged oral maintenance therapy. Eighty-eight percent of patients entered complete remission. With a median follow-up of 77 months (range, 48 to 111 months), 42% +/- 6% (SEM) of patients achieving remission are projected to remain disease-free at 5 years, and disease-free survival for all patients entered on study is 35% +/- 5%. Failure to achieve remission within the first 4 weeks of therapy and the presence of the Philadelphia chromosome are associated with a 100% risk of relapse. Remission patients with neither of these adverse features have a 48% +/- 6% probability of remaining in continuous remission for 5 years. Patients with T-cell phenotype have a favorable prognosis with 59% +/- 13% of patients achieving remission remaining disease-free compared with 31% +/- 7% of CALLA-positive patients. Intensive chemotherapy may produce prolonged disease-free survival in a sizable fraction of adults with ALL. Improved therapy is needed, especially for patients with adverse prognostic features.

Adult↗
PubMed1991

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.

Adenosine Triphosphatases↗
PubMed1991

Constitution of the twin polymerase of DNA polymerase III holoenzyme.

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.

Chromatography, Gel↗
PubMed1991

Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme.

DNA polymerase III holoenzyme (holoenzyme), the multiprotein replicase of Escherichia coli, is essentially unlimited in processive DNA synthesis. Processive activity can be reconstituted from two components. One component, the beta preinitiation complex, is a beta dimer clamped onto primed DNA. The beta preinitiation complex is formed by the five-protein gamma complex, which hydrolyzes ATP to chaperone beta onto primed DNA. The other component is the alpha epsilon polymerase. The alpha epsilon polymerase itself is not processive, but is endowed with extremely high processive activity upon assembly with the beta preinitiation complex. Here we examine the mechanism by which the beta preinitiation complex confers processivity onto the alpha epsilon polymerase. We find the beta preinitiation complex to be mobile on DNA. Diffusion of beta on DNA is specific to duplex DNA, is bidirectional, does not require ATP, and appears to diffuse linearly along the duplex. Furthermore, beta directly binds the alpha epsilon polymerase through contact with alpha, the DNA polymerase subunit. Hence, the high processivity of the holoenzyme is rooted in a "sliding clamp" of beta on DNA that tethers the polymerase to the primed template. Implications for transcription and translation are discussed.

DNA Polymerase III↗