Biomedical subjects

J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 73 records · Page 4Linked to original sources

PubMed1996

In vitro reconstitution of human replication factor C from its five subunits.

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.

DNA Replication↗

PubMed1996

A nucleolar RNA helicase recognized by autoimmune antibodies from a patient with watermelon stomach disease.

Watermelon stomach is characterized by prominent stripes of ectatic vascular tissue in the stomach similar to stripes on a watermelon; in patients with this disorder chronic gastrointestinal bleeding occurs and approximately half of these patients have associated autoimmune disorders. In the serum of one patient, an antinucleolar antibody titer of 1:25 600 was found; the antibodies specifically recognized an approximately 100 kDa nucleolar protein, which we referred to as the 'Gu' protein. Its cDNA was cloned and sequenced. The Gu protein is a member of a new subgroup of RNA helicases, the DEXD box family. Gu protein fused with glutathione S-transferase contains ATP-dependent RNA helicase activity which preferably translocates in the 5'-->3' direction. Its RNA folding activity, RNA-dependent ATPase and dATPase activities, and its translocation direction are similar to those of RNA helicase II [Flores-Rozas, H. and Hurwitz, J. (1993) J. Biol. Chem. 268, 21372-21383]. Sequencing of 209 amino acids of RNA helicase II peptides showed 96.7% identity with the cDNA-derived amino acid sequence of the Gu protein. The precise biological roles of this RNA helicase in the biogenesis of ribosomal RNA and the pathogenesis of watermelon disease and autoimmune disorder require further study.

Adenosine Triphosphatases↗

PubMed1996

Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

Amino Acid Sequence↗

PubMed1996

Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases.

BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.

Bacteriophage T4↗

PubMed1995

Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21.

p21, a p53-induced gene product that blocks cell cycle progression at the G1 phase, interacts with both cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor for DNA polymerases delta and epsilon and is required for both DNA replication and nucleotide excision repair. Previous studies have shown that p21 inhibits simian virus 40 (SV40) DNA replication in HeLa cell extracts by interacting with PCNA. In this report we show that p21 blocks nucleotide excision repair of DNA that has been damaged by either ultraviolet radiation or alkylating agents, and that this inhibition can be reversed following addition of PCNA. We have determined that p21 is more effective in blocking DNA resynthesis than in inhibiting the excision step. We further show that a peptide derived from the carboxyl terminus of p21, which specifically interacts with PCNA, inhibits polymerase delta-catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA. When added at higher levels, this peptide also blocks both SV40 DNA replication and nucleotide excision repair in HeLa cell extracts. These results indicate that p21 interferes with the function of PCNA in both in vitro DNA replication and nucleotide excision repair.

Amino Acid Sequence↗

PubMed1995

Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex.

The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.

Adenosine Triphosphate↗

PubMed1995

Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1.

Mammalian cell-cycle control by antimitogenic signals involves p21Cip1/WAF1 (refs 1-4), p27Kip1 (refs 5, 6) and p57Kip2 (refs 7, 8), a family of proteins that bind to and inhibit cyclin-dependent kinases (CDKs) required for initiation of S phase. The protein p21 also binds to the DNA polymerase delta processivity factor, proliferating-cell nuclear antigen (PCNA), and inhibits in vitro PCNA-dependent DNA replication. The CDK and PCNA inhibitory activities of p21 are shown here to be functionally independent and to reside in separate protein domains. The PCNA binding and inhibitory activities, which are not observed with p27 or p57, reside in the C-terminal domain of p21, whereas the CDK inhibitory activity resides in the conserved N-terminal domains of these proteins. When separately overexpressed in mammalian cells, the CDK and PCNA inhibitory domains prevent DNA replication, demonstrating a dual function of p21 as a cell-cycle inhibitor in vivo.

Amino Acid Sequence↗

PubMed1995

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

DNA Repair↗

PubMed1995

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23.

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Base Sequence↗

PubMed1994

The bovine papillomavirus E2 protein modulates the assembly of but is not stably maintained in a replication-competent multimeric E1-replication origin complex.

Initiation of bovine papillomavirus (BPV) DNA synthesis in vivo and in vitro depends on the interaction of the viral initiator protein E1 with the replication origin (ori+ DNA). The viral E2 protein assists this interaction, resulting in a cooperative assembly of both proteins on the replication origin. Using gel mobility-shift experiments, we demonstrate that in the presence of both E1 and E2 proteins two classes of ori+ DNA complexes were formed: complex 1 (c1) and complex 2 (c2). Formation of c1 depended on both the E1 and E2 proteins and both proteins were contained within c1. The generation of c2 was dependent on the E1 protein and could be enhanced by E2, but the E2 protein was not detected within c2. At high E2/E1 ratios, c1 was the dominant complex formed. Under these conditions, E1-dependent BPV DNA synthesis in vitro was inhibited. At low E2/E1 ratios, the stimulation of c2 was correlated with the stimulation of BPV DNA replication by E2 in vitro. These data suggest that E2 assists E1 in the formation of an intermediate c1 complex, which is replication inactive. The c1 complex is converted in turn to the replication-active c2 complex, which contains E1 but lacks E2. We propose that the ratios of c1 and c2 formed in response to the levels of E1 and E2 protein determine the potential for BPV DNA synthesis in vitro and in vivo and may contribute to copy number regulation of BPV plasmids within the cell.

Base Sequence↗

PubMed1994

Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.

CDC2-CDC28 Kinases↗

PubMed1994

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.

CDC2-CDC28 Kinases↗

PubMed1994

Replication of bovine papillomavirus type 1 origin-containing DNA in crude extracts and with purified proteins.

The in vitro replication of DNA containing the bovine papillomavirus (BPV-1) origin has been carried out with cell-free extracts from mouse FM3A and human HeLa cells. DNA synthesis required the E1 protein, the minimal origin of replication (nucleotides 7911-22 of the BPV-1 genome), and, at low levels of FM3A extract, the addition of the human single-stranded DNA-binding protein (also called RP-A or RF-A). The E2 protein was not absolutely required, but could stimulate DNA synthesis at low levels of E1. DNA synthesis was also reconstituted using purified proteins from HeLa cells. These protein factors included human single-stranded DNA-binding protein, topoisomerase I, and DNA polymerase (pol) alpha-primase complex. At low concentrations of pol alpha-primase complex, the formation of high molecular weight products was dependent on the addition of DNA polymerase delta holoenzyme containing proliferating cell nuclear antigen and activator 1, also called RF-C. We have overexpressed and isolated the E1 protein from bacteria. This protein also supported BPV DNA synthesis, both in crude extracts and with purified proteins suggesting that E1 phosphorylation is not required for BPV DNA replication in vitro.

Animals↗

PubMed1994

Initiation of DNA replication by simian virus 40 T antigen is inhibited by the p107 protein.

The p107 protein is related to the Rb protein by a 60-kDa region of homology called the pocket domain that binds cellular proteins such as E2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T antigen-mediated replication of SV40 DNA in vitro. The pocket domain inhibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins formed a ternary complex with DNA containing T antigen-binding site I. The pocket domain of p107 did not inhibit the oligomerization of T antigen in the absence of DNA, and the p107 derivative is bound to the intermediates of this reaction. In the unwinding assay, once T antigen was preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the pocket domain bind to a synthetic replication fork, and this complex supported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a partial duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in HeLa cell crude extracts or in the monopolymerase reaction. This inhibition is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively regulate T antigen-mediated replication in vitro. The binding of T antigen to p107 is discussed with respect to its role in mitigating negative cell growth control, resulting in viral mediated transformation.

Animals↗

PubMed1994

Reconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses.

Human single-stranded DNA-binding protein (HSSB), also known as replication protein A, is composed of a 70-kDa single-stranded DNA-binding subunit (p70) and 34-kDa and 11-kDa (p34 and p11, respectively) subunits of unknown functions. We have examined interactions among the HSSB subunits in vivo by coinfecting insect cells with different combinations of recombinant baculoviruses encoding p70, p34, or p11. In vivo, coexpressed p34 and p11 subunits formed stable complexes, whereas neither p34 nor p11 formed stable complexes with p70. In cells coinfected with viruses expressing all three subunits, the stable heterotrimer formed, which, when purified, replaced HSSB isolated from HeLa cells in various assays, including simian virus 40 DNA replication in vitro. These data suggest that, in the assembly of functionally active HSSB, formation of the p34-p11 complex precedes p70 addition to the complex.

Animals↗

PubMed1994

Diagnosis of parathyroid adenomas: efficacy of measuring parathormone levels in needle aspirates of cervical masses.

OBJECTIVE: The purpose of this study was to assess the value of measuring parathormone levels in percutaneous needle aspirates of suspicious cervical lesions in patients with hyperparathyroidism to confirm whether the lesion represents abnormal parathyroid tissue. SUBJECTS AND METHODS: The study group consisted of 66 patients with hyperparathyroidism in whom 80 cervical lesions were aspirated and levels of parathormone in the aspirates were measured. CT guidance was used for two patients and sonographic guidance for the remainder. The lesions selected for aspiration were demonstrated on either sonography or CT and had either an unusual position (separate from the thyroid gland or were intrathyroidal) or configuration (irregular shape or atypical heterogeneous sonographic texture). In 15 patients, an indeterminate, posteriorly located intrathyroidal mass was detected and felt most likely to represent a thyroid nodule by sonographic criteria. These masses were aspirated to rule out atypical parathyroid adenomas. In patients who had been previously explored for hyperparathyroidism and presented with persistent or recurrent hypercalcemia, all indeterminate, cervical, potentially parathyroid masses were aspirated for parathormone determination. The level of parathormone in each aspirate was measured by using an immunoradiometric assay. RESULTS: Levels of parathormone were increased in the aspirates in 37 of the 45 patients in whom sonography showed classic lesions suggestive of parathyroid adenoma. This included the 25 patients who had previously undergone exploratory surgery. At surgery, all 37 had parathyroid adenomas in the indicated locations, for a specificity of 100%. In six patients, the results of the aspiration were false-negative. Parathyroid adenomas were suspected on sonograms and confirmed at surgery, but no parathormone was detected in the aspirate. Results of aspiration of indeterminate lesions were true-negative in two patients who had both characteristic and indeterminate lesions on sonography and in the 15 patients who had indeterminate lesions that were felt to be of thyroid origin. Aspirates contained no parathormone, and surgical findings confirmed the lesions were not of parathyroid origin. CONCLUSION: Our results show that increased levels of parathormone in percutaneous needle aspirates of cervical masses in patients with hyperparathyroidism confirm the mass is a parathyroid adenoma. Although absence or low levels of parathormone in the aspirates usually excludes a parathyroid adenoma, this is not absolute as sometimes the needle may miss the mass, rendering the parathormone value invalid (false-negative).

Adenoma↗

PubMed1993

Characterization of a new RNA helicase from nuclear extracts of HeLa cells which translocates in the 5' to 3' direction.

RNA helicase II, isolated from nuclear extracts of HeLa cells, was purified 1,300-fold and contained a single protein band of 100 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displaced partial duplex RNA exclusively in a 5' to 3' direction. This reaction was supported only by ATP and deoxy-ATP at relatively high concentrations (the Km was estimated as 1 mM). The enzyme displayed only ATPase and deoxy-ATPase activity that was stimulated preferentially by poly(C). RNA helicase catalyzed the unwinding of duplex RNA and RNA.DNA hybrids provided that single-stranded (ss) RNA was available for the helicase to bind. In the presence of MgCl2 and ATP or adenosine 5'-O-(3-thiotriphosphate) RNA helicase II interacted with ssRNA and yielded a protein-RNA complex only when reaction mixtures were treated with glutaraldehyde after incubation. When the reactions contained nonhydrolyzable ATP analogs or GTP, ssRNA was converted into an electrophoretically slower migrating form by the helicase. The slower migrating RNA form was shown to be an RNA species containing secondary structure that resided within a putative hairpin loop. These observations indicate that RNA helicase II can introduce intramolecular secondary structure in ssRNA.

Adenosine Triphosphatases↗

PubMed1993

Reconstitution of cyclin-dependent cdc2 and cdk2 kinase activities in vitro.

The genes that encode human cdc2 and cdk2 proteins are essential for cell cycle progression. In this report, we describe the purification of cyclin-associated cdc2 and cdk2 kinases as well as cyclin-free cdc2 and cdk2 protein preparations from HeLa cells. The cdc2-cyclin B kinase complex that we have isolated, consisting of two polypeptides of p60 (cyclin B) and p34 (cdc2), phosphorylated both the p34 and p70 subunits of the three-subunit human single-stranded DNA-binding protein (also called RP-A), a DNA replication and repair factor. We also partially purified a histone H1 kinase activity that is associated with the cdk2 and cyclin A proteins. Purified human cyclins A and B1, overproduced in bacteria, complemented a cellular fraction enriched in cdc2 and cdk2 proteins to reconstitute histone H1 kinase activity. Using this complementation system, human cdc2 and cdk2 proteins were purified and separated from one another. Glycerol gradient analyses demonstrated that the purified cdk2 (p33) protein co-sedimented with a cyclin A-dependent H1 kinase activity. Thus, cdk2 and cyclin A proteins are components that assemble to yield a kinase complex that catalyzes the phosphorylation of histone H1.

CDC2 Protein Kinase↗