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J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 91 records · Page 5Linked to original sources
PubMed1993

Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities.

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.

CDC2 Protein Kinase↗
PubMed1993

Human RNA helicase A is homologous to the maleless protein of Drosophila.

RNA helicase A is an abundant nuclear enzyme of HeLa cells that unwinds double-stranded RNA in a 3' to 5'direction (Lee, C. G., and Hurwitz, J. (1992) J. Biol. Chem. 267, 4398-4407). A complementary DNA (cDNA) clone expressing RNA helicase A was isolated by screening a human cDNA library with polyclonal antibodies produced against the purified protein. The deduced amino acid sequence from this clone showed that RNA helicase A is a member of the DEAH family of proteins thought to be helicases. Sequence comparison among all known proteins of the DEAH family revealed that the highest homology was between RNA helicase A and the maleless protein (MLE) of Drosophila. There was 49% identity and 85% similarity throughout the overall primary sequences of both proteins, suggesting that RNA helicase A is the human counterpart of Drosophila MLE. Polyclonal antibodies against Drosophila MLE recognized RNA helicase A in crude nuclear extracts of HeLa cells as well as the purified protein. A recombinant RNA helicase A containing 6 histidine residues at the NH2 terminus was expressed in Sf9 cells using a baculovirus vector. The protein isolated from insect cells and the enzyme purified from HeLa cells exhibited identical RNA helicase and RNA-dependent ATPase activities.

Amino Acid Sequence↗
PubMed1993

Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site.

Using quantitative gel retardation assays the properties of the bovine papilloma virus (BPV) origin recognition protein E1 and the effect of the viral E2 protein on the binding of E1 to BPV origin DNA were examined. As reported previously (Seo, Y.S., Mueller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. and J. Hurwitz (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2865-2869), the E1 protein binds specifically to DNA sequences within the BPV origin (ori+) of replication. We also show that the presence of MgCl2 and ATP could stabilize the E1 ori+ DNA complex. At low levels of E1, ori+ DNA binding was greatly stimulated by the viral E2 protein when the intact E2 binding site 12 was present on the DNA. In addition DNA-protein complexes formed in the presence of both E1 and E2 were more stable than those formed with E1 alone. In the absence of an E2 binding site the E2 protein inhibited the binding of E1 to the BPV origin. Spacing of 0 or 9 base pairs between the E1 binding site and the E2 binding site 12 abolished the stimulation of E1-DNA binding by E2, whereas spacing of 6 base pairs between the two binding sites allowed for efficient stimulation. The data presented account for a direct role of E2 in BPV DNA replication. We propose that the cooperative binding of both the E1 and E2 proteins to BPV ori+ DNA is mediated by protein-protein interactions and by protein-DNA interactions, which include the formation of specific contacts of E2 with DNA.

Adenosine Triphosphate↗
PubMed1993

RNA annealing activity is intrinsically associated with U2AF.

U2AF is a protein that is essential for the formation of the prespliceosome complex during pre-mRNA splicing. It contains two subunits, 65 and 35 kDa, although only the 65-kDa subunit has been shown to be essential for its splicing activity. Here, we show that the 65-kDa subunit mediates the annealing of complementary single-stranded RNAs or single-stranded DNAs. This activity was shown to reverse the action of RNA helicase A, an enzyme that catalyzes the displacement of duplex RNAs. The NH2-terminal region of the 65-kDa subunit of U2AF, containing arginine-serine (RS) dipeptides and basic amino acid sequences, was shown to be essential for the annealing of complementary sequences, RNA binding, and the inhibition of RNA helicase A activity. Thus, through the combined action of U2AF and RNA helicases, duplex RNA regions can be reversibly formed and displaced. Such reactions appear to be critical for pre-mRNA splicing, translation, and transcription.

Base Sequence↗
PubMed1993

Functional interactions between SV40 T antigen and other replication proteins at the replication fork.

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen.

Antigens, Polyomavirus Transforming↗
PubMed1993

DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication.

The ATP-dependent binding of simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication is an essential step in the initiation of SV40 DNA synthesis. Previous studies indicated that the ATP-dependent complex consists of a double hexamer of T antigen at the origin. The binding reaction and the subsequent unwinding of the duplex DNA from the origin were examined using a gel mobility shift assay. T antigen bound to the core origin cooperatively in the presence of ATP. In the presence of human single-stranded DNA-binding protein (HSSB), T antigen, complexed to the core origin, started the unwinding of duplex DNA. At low concentrations of T antigen and in the presence of ATP, DNA polymerase alpha (pol alpha) stimulated the binding of T antigen to the core origin, while HSSB did not. This stimulation resulted in an increase in the subsequent unwinding reaction in the presence of HSSB. Primase alone did not affect the binding reaction and was not required for the stimulation by pol alpha. The stimulation required the hydrolysis of ATP and the AT tract domain of the core origin. Kinetic studies showed that while pol alpha stimulated the binding of T antigen to the core origin, it did not stabilize the complex. Pol alpha also stimulated the formation of the ATP-dependent T antigen-site I complex, a region that also contains an AT-rich sequence. These observations imply a regulatory role for pol alpha in the initiation of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed1993

Isolation of helicase alpha, a DNA helicase from HeLa cells stimulated by a fork structure and signal-stranded DNA-binding proteins.

A DNA helicase, called DNA helicase alpha, was purified from HeLa cells to apparent homogeneity. The helicase and its single-stranded DNA-dependent ATPase activities cosedimented in glycerol gradients with two polypeptides of 110 and 90 kDa with a sedimentation coefficient of 7.4 S. The DNA helicase was markedly stimulated by DNA substrates with a 5'-tailed fork. A DNA substrate with a 3'-tailed fork structure was less stimulatory, although it was more active than substrates without a fork. The directionality of unwinding is 3'-->5' with respect to the single-stranded DNA to which the enzyme was bound. The helicase activity also required a single-stranded DNA-binding protein (SSB) for unwinding activity. The stimulation by SSBs was nonspecific; all SSBs tested, such as human SSB, bacteriophage T4 gene 32, and Escherichia coli SSB, stimulated the DNA helicase activity to a varying extent in the presence of a fork structure. With long duplex substrates (> 500 base pairs), the presence of a fork substantially stimulated the DNA helicase activity in the presence of E. coli SSB. Human SSB stimulated the DNA helicase activity to the greatest extent (> 10-fold) with a substrate containing a fork compared with substrates without a fork. DNA helicase activity required ATP hydrolysis and could be supported by all eight nucleoside triphosphates. The Km values for ATP and dATP in unwinding were 28 and 48 microM, respectively. In general, ribonucleoside triphosphates were better effectors than deoxyribonucleoside triphosphates. The properties of this DNA helicase make it a candidate for a DNA replicative helicase in human cells.

Adenosine Triphosphatases↗
PubMed1993

Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin.

The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, E1 and E2, while all other proteins are derived from the host. We described previously the isolation of the E1 protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The E1 protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maximal efficiency. The E1 protein also catalyzed a BPV origin-dependent DNA unwinding reaction. In this report, we show that at low levels of E1 protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by E1, but it had no effect on the nonspecific E1-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the E1 protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing E1 binding to the BPV origin via DNA-protein and protein-protein interactions.

Animals↗
PubMed1993

Bovine papilloma virus (BPV)-encoded E1 protein contains multiple activities required for BPV DNA replication.

Replication of bovine papilloma virus (BPV) DNA requires two virus-encoded proteins, E1 and E2, while all other proteins are supplied by the host cell. Here, we describe the isolation of the E1 protein and show that it is a multifunctional protein. Purified E1 protein was required for the in vitro replication of BPV origin-containing DNA by extracts of mouse cells, as reported [Yang, L., Li, R., Mohr, I. J., Clark, R. & Botchan, M. R. (1991) Nature (London) 353, 628-632]. In addition, the E1 protein cosedimented with a number of other activities including (i) DNA helicase activity, (ii) BPV origin-containing DNA-specific binding activity, (iii) DNA-dependent ATPase activity, and (iv) BPV origin-specific unwinding of superhelical DNA. The E1 protein, acting as a helicase, moved in the 3'-->5' direction, like simian virus 40 (SV40) large tumor antigen, which plays a pivotal role in SV40 DNA replication. However, unlike the SV40 large tumor antigen, the helicase activity of E1 was stimulated 5-fold by the presence of a fork structure at the junction between single-stranded and double-stranded DNA and was supported efficiently by all eight nucleoside triphosphates. The E1-catalyzed ATPase activity required the presence of single-stranded or double-stranded DNAs.

Adenosine Triphosphatases↗
PubMed1993

The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis.

p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.

Autoantigens↗
PubMed1993

Mapping of the 70 kDa, 34 kDa, and 11 kDa subunit genes of the human multimeric single-stranded DNA binding protein (hSSB/RPA) to chromosome bands 17p13, 1p35-p36.1, and 7p21-p22.

Human single-stranded DNA binding protein (hSSB/RPA) is a multimeric single-stranded DNA binding protein consisting of three subunits of 70 kDa, 34 kDa, and 11 kDa. Human SSB was isolated from HeLa cells as an essential factor for the in vitro replication of simian virus 40 DNA. We and others have isolated and sequenced cDNAs for each subunit of the SSB. The chromosome on which each gene is located was determined through the analysis of a panel of human/hamster somatic cell hybrids using the polymerase chain reaction with pairs of synthetic oligonucleotide primers from the 3'-untranslated sequences of the genes. Genomic clones for each gene were isolated from a genomic cosmid library prepared from human lymphoblastoid cells. Using those clones as probes, we have carried out fluorescence in situ hybridization to human metaphase chromosomes and have mapped the 70 kDa subunit gene to 17p13, the 34 kDa subunit gene to 1p35-p36.1, and the 11 kDa subunit gene to 7p21-p22. Since hSSB participates in replication, recombination and repair of DNA, the physical mapping of hSSB genes may aid in the identification of human hereditary diseases associated with aberrant DNA reactions caused by genetic alterations of the hSSB.

Animals↗
PubMed1992

Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein-terB complex of Escherichia coli.

The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined. In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems. With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient. These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner. We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence. The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen. With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E. coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation. However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked. Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.

Antigens, Polyomavirus Transforming↗
PubMed1992

The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.

Adenosine Triphosphate↗
PubMed1992

Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells.

The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C). A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa). In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells. This gene was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP. Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E. coli DNA polymerase III holoenzyme. Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction. Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction. When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone. These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process.

Amino Acid Sequence↗
PubMed1992

The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems.

The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.

Antibodies, Monoclonal↗
PubMed1992

Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells.

Activator 1 (A1; also called replication factor C), in conjunction with proliferating-cell nuclear antigen (PCNA), is essential for the elongation of primed DNA templates by DNA polymerases delta and epsilon. A1 contains five distinct subunits of 145, 40, 38, 37, and 36.5 kDa. Here we describe the isolation, sequence, and bacterial expression of a cDNA coding for the 40-kDa subunit. In keeping with the presence of an ATP-binding motif, the bacterially expressed 40-kDa subunit binds ATP. The interaction between the 40-kDa subunit and ATP was reduced by the addition of PCNA. In addition, antibodies raised against the 40-kDa subunit abolished the A1- and PCNA-dependent synthesis of DNA catalyzed by polymerase delta. The putative amino acid sequence of the 40-kDa subunit of A1 revealed significantly homology with the bacteriophage T4 gene 44 protein and, to a lesser degree, with the tau and gamma subunits of Escherichia coli DNA polymerase III holoenzyme.

Adenosine Triphosphate↗