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Biomedical subjects

M O'Donnell

Publications and source records attributed to M O'Donnell.

At least 91 records · Page 5Linked to original sources
PubMed1999

UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V.

The damage-inducible UmuD' and UmuC proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD'(2)C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD'(2)C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD'(2)C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, beta sliding clamp, gamma clamp loading complex, and E. coli single-stranded binding protein (SSB), pol V's polymerase activity is highly "error prone" at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the "A-rule," dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD'(2)C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3'-OH template-primer ends. We show, however, that despite competition for primer-3'-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.

Base Sequence↗
PubMed1999

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or epsilon)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

Amino Acid Substitution↗
PubMed1999

The internal workings of a DNA polymerase clamp-loading machine.

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.

Adenosine Triphosphate↗
PubMed1999

Trading places on DNA--a three-point switch underlies primer handoff from primase to the replicative DNA polymerase.

This study reports a primase-to-polymerase switch in E. coli that closely links primase action with extension by DNA polymerase III holoenzyme. We find that primase tightly grips its RNA primer, protecting it from the action of other proteins. However, primase must be displaced before the beta sliding clamp can be assembled on the primed site. A single subunit of the holoenzyme, chi, is dedicated to this primase displacement task. The displacement mechanism depends on a third protein, SSB. Primase requires contact to SSB for its grip on the primed site. The chi subunit also binds SSB, upon which the primase-to-SSB contact is destabilized leading to dissociation of primase and assembly of beta onto the RNA primer. The conservation of this three-point switch, in which two proteins exchange places on DNA via mutually exclusive interaction with a third protein, is discussed.

DNA↗
PubMed1999

The effect of vomitoxin (Deoxnivalenol) on testicular morphology, testicular spermatid counts and epididymal sperm counts in IL-6KO [B6129-IL6 [TmlKopf] (IL-6 gene deficient)] and WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] mice.

The potential of vomitoxin (VT) to affect testicular morphology and testicular and epididymal sperm counts was assessed in three strains of mice: IL-6KO [B6129-IL6 (tmlKopf) (IL-6 gene deficient)], WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] and B6C3F1 mice in a 90-day feeding study. The treated mice received VT at a concentration of 10 ppm in their diet. The body weight of VT-treated animals was significantly reduced compared with control animals. Slight changes, not statistically significant, were observed in relative testis weight and testicular spermatid counts. Histological changes were not apparent in the testes of VT-treated animals. The diameter of the seminiferous tubules, the height of the seminiferous epithelium and the number of Sertoli cell nucleoli per cross-sectioned seminiferous tubule in the VT-treated groups were not significantly different from their respective untreated controls. The IL-6KO and B6C3F1 VT-treated mice had significantly reduced cauda epididymal weights compared with their respective controls. These changes were not attributed to decreased sperm counts and this finding suggests that VT may exert an adverse affect on the epididymis.

Animals↗
PubMed1999

Replisome assembly at oriC, the replication origin of E. coli, reveals an explanation for initiation sites outside an origin.

This study outlines the events downstream of origin unwinding by DnaA, leading to assembly of two replication forks at the E. coli origin, oriC. We show that two hexamers of DnaB assemble onto the opposing strands of the resulting bubble, expanding it further, yet helicase action is not required. Primase cannot act until the helicases move 65 nucleotides or more. Once primers are formed, two molecules of the large DNA polymerase III holoenzyme machinery assemble into the bubble, forming two replication forks. Primer locations are heterogeneous; some are even outside oriC. This observation generalizes to many systems, prokaryotic and eukaryotic. Heterogeneous initiation sites are likely explained by primase functioning with a moving helicase target.

Bacterial Proteins↗
PubMed1999

Biliary obstruction in hematopoietic cell transplant recipients: an uncommon diagnosis with specific causes.

Jaundice is a common problem in marrow transplant recipients. The incidence of bile duct obstruction in this setting is unknown. The purpose of this study was to determine the incidence of biliary obstruction, the causes, and outcomes following marrow transplant. Consecutive cases were reviewed at two major transplant centers in the United States from 1969 to 1996 at the Fred Hutchinson Cancer Research Center and 1989 to 1996 at the City of Hope National Medical Center. Nine cases of biliary obstruction were identified as a cause of jaundice in 7412 marrow transplant recipients, an incidence of 0.12%. The presentation was bimodal, with seven cases occurring prior to day 100 and two occurring 2 to 4 years after transplantation. The age distribution was 15 to 50 years and all patients had received allogeneic transplants. The causes of obstruction included gallbladder sludge (n=1), a duodenal hematoma (n=1), choledocholithiasis with biliary pancreatitis (n=1), bile duct infection (n=2), recurrent malignancy (n=1), choledocholithiasis associated with a benign stricture (n=1), Epstein-Barr virus-related lymphoproliferative disorder (n=1), and a benign stricture of unknown etiology (n=1). Biliary obstruction is a rare cause of jaundice in the post-transplant period. The presentation was similar to that of other post-transplant hepatobiliary problems, but with disparate causes.

Adolescent↗
PubMed1999

A new bipolar diathermy probe for the outpatient management of adult acute epistaxis.

The findings of a prospective evaluation of the use of a newly-designed bipolar diathermy probe in the outpatient management of adult acute epistaxis are presented. Forty-four adult patients with acute epistaxis were treated with bipolar diathermy after identification of the bleeding point using a rod lens endoscope. The use of the nasal endoscope allowed diagnosis of the source of the bleeding point in all patients. In all 44 patients immediate control of the bleeding was achieved. The use of the bipolar probe obviated the need for hospital admission in 31 patients (70%). Three patients had recurrence of their epistaxis after treatment (7%). The number of patients requiring admission to hospital with epistaxis was significantly reduced using the new bipolar strategy. (4.4/month versus 1.2/month, P < 0.002)

Acute Disease↗
PubMed1999

Poorly differentiated adenocarcinoma with extensive rhabdoid differentiation: clinicopathological features of two cases arising in the gastrointestinal tract.

This paper describes the pathological features of two cases of composite extra-renal rhabdoid tumor; that is, poorly differentiated carcinoma with extensive rhabdoid differentiation, arising in the small intestine of a 64-year-old man and in the pancreas of a 77-year-old woman. In both cases the tumor behaved aggressively and the patients died from disseminated malignancy within 3 months of original presentation and diagnosis. To the best of our knowledge these are the first examples of such tumors in the small intestine and pancreas.

Adenocarcinoma↗
PubMed1999

A study of client-focused case management and consumer advocacy: the Community and Consumer Service Project.

OBJECTIVE: The study investigated the provision of client-focused services to community-based clients with schizophrenia and bipolar disorder. It hypothesised that the delivery of more client-focused services would improve client outcome in terms of functioning, disability and satisfaction with services. Client-focused services were developed using an empowerment model of case management and by the addition of consumer advocates. METHOD: Clients referred for case management were randomly allocated to one of three groups: standard case management (n = 35), client-focused case management (n = 39), or client-focused case management plus consumer advocacy (n = 45). Measures of functioning, disability, quality of life, burden of care and service satisfaction were measured at baseline and 12 months. Outcome data were collected concerning number and duration of hospital readmission, crisis intervention and compliance with treatment and services. RESULTS: While there were no differences between the groups on quantitative measures of functioning, disability, quality of life, service satisfaction and burden of care, there were significant between-group differences on qualitative measures of satisfaction with services. CONCLUSIONS: Several methodological difficulties hampered interpretation of the findings. Although clients did not differ on outcome measures of functioning and disability, the group receiving client-focused case management reported greater satisfaction with service delivery.

Adolescent↗
PubMed1999

Determinants of the excessive rates of lung cancer in northeast Florida.

BACKGROUND: Age-adjusted rates of lung cancer among whites, but not blacks, in Duval County Jacksonville), Florida, have been among the highest of any metropolitan area of the United States for three decades. METHODS: We conducted a case-control study, interviewing 507 patients with lung cancer diagnosed from 1993 to 1996 and 1,007 control subjects, randomly selected from the county population. RESULTS: Cigarette smoking was the dominant cause of lung cancer (odds ratios, 59 among male current smokers; 30 among female current smokers). Both prevalence and intensity of smoking were excessive for whites but less for blacks when compared with national norms. Little association was found with residential patterns, indices of air quality, or occupation. Risk doubled with increased intake of dietary fat and with a family history of lung cancer. CONCLUSIONS: Life-style factors, especially smoking, but not environmental factors, appear responsible for the high rates of lung cancer in northeast Florida.

Adult↗
PubMed1998

cDNA cloning and gene mapping of human homologs for Schizosaccharomyces pombe rad17, rad1, and hus1 and cloning of homologs from mouse, Caenorhabditis elegans, and Drosophila melanogaster.

Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy. The Schizosaccharomyces pombe genes rad17, rad1, and hus1 have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways. We have cloned the cDNA for the human homolog of S. pombe rad17, RAD17, which localizes to chromosomal location 5q13 by fluorescence in situ hybridization and radiation hybrid mapping; the cDNA for the human homolog of S. pombe rad1, RAD1, which maps to 5p14-p13.2; and the cDNA for the human homolog of S. pombe hus1, HUS1, which maps to 7p13-p12. The human gene loci have previously been identified as regions containing tumor suppressor genes. In addition, we report the cloning of the cDNAs for genes related to S. pombe rad17, rad9, rad1, and hus1 from mouse, Caenorhabditis elegans, and Drosophila melanogaster. These include Rad17 and Rad9 from D. melanogaster, hpr-17 and hpr-1 from C. elegans, and RAD1 and HUS1 from mouse. The identification of homologs of the S. pombe rad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes.

Amino Acid Sequence↗
PubMed1998

ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits.

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.

Adenosine Triphosphate↗
PubMed1998

ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme.

The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the gamma complex during clamp loading. We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway. The gamma complex (gamma2 delta delta'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-2. 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M). Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation. This ATP-bound gamma complex binds beta and opens the ring at the dimer interface. Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex. open beta ring.DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.

Adenosine Triphosphate↗
PubMed1998

Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA.

The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta. Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.

Adenosine Triphosphate↗
PubMed1998

Biochemical basis of SOS-induced mutagenesis in Escherichia coli: reconstitution of in vitro lesion bypass dependent on the UmuD'2C mutagenic complex and RecA protein.

Damage-induced SOS mutagenesis requiring the UmuD'C proteins occurs as part of the cells' global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD'2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD'2C, activated RecA protein (RecA*), beta-sliding clamp, gamma-clamp loading complex, single-stranded binding protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD'2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD'2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.

Bacterial Proteins↗