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Biomedical subjects

J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 109 records · Page 6Linked to original sources
PubMed1992

A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction.

We have purified an RNA helicase to near homogeneity from nuclear extracts of HeLa cells. The enzyme migrated as a 130-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and exhibited a sedimentation coefficient of 6.4 on glycerol gradient centrifugation. The enzyme translocated in a 3' to 5' direction and acted catalytically, displacing at least a 4-fold molar excess of duplex RNA compared with the enzyme added. All eight common nucleoside triphosphates supported RNA helicase activity at relatively low concentrations (Km in values in the 15-20 microM level). In the presence of RNA and some single-stranded DNAs, the RNA helicase hydrolyzed all nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate. The enzyme displaced deoxyribooligonucleotides provided they were hydrogen-bonded to RNA possessing 3' single-stranded regions, but it did not displace ribooligonucleotides hydrogen-bonded to DNA containing 3' single-stranded regions. The enzyme, in the absence of ATP, binds to both single-stranded RNA and DNA, but the amount of complex formed with RNA was 20-fold greater than the complex formed with DNA. In both cases, the complex formed in the absence of ATP was rapidly reversed by the addition of ATP and not by adenyl-5'-yl (beta,gamma-methylene)-diphosphate. We propose that the enzyme can bind to both single-stranded RNA and DNA and hydrolyze ATP, but by virtue of its greater stability on RNA, the enzyme can only translocate on RNA possessing 3' single-stranded regions.

Adenosine Triphosphate↗
PubMed1992

Nonconservative segregation of parental nucleosomes during simian virus 40 chromosome replication in vitro.

Simian virus 40 chromosomes can be replicated in vitro with the same set of purified proteins required for the replication of naked DNA containing the viral origin. With these reconstituted systems, the fate of parental histones during replication was examined in vitro. The assembly of nucleosomes on replicating chromosomes was hardly affected by the presence of simultaneously replicating naked DNA competitor, suggesting that replication forks can traverse nucleosomes without the displacement of histones. Moreover, we demonstrate that the nascent nucleosomes were distributed almost equally between the leading and lagging strands. This distributive mode of nucleosome segregation favors the propagation of parental chromatin structures to both daughter cells, which can maintain cellular functions dictated by these structures during cell proliferation.

DNA Polymerase II↗
PubMed1992

Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation.

DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase alpha (pol-alpha), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase delta, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-alpha extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-alpha, conditions under which pol-alpha was markedly inhibited. These findings suggest that interactions between T antigen, pol-alpha-primase, and HSSB position the pol-alpha-primase complex on the lagging-strand template for RNA primer synthesis.

Antigens, Polyomavirus Transforming↗
PubMed1992

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.

DNA Replication↗
PubMed1991

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

Autoantigens↗
PubMed1991

Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity.

A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells. At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity. At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity. Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA. The addition of E. coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase. One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase. Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity. The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction. The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment. Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.

Adenosine Triphosphatases↗
PubMed1991

The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells.

An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).

Adenosine Triphosphate↗
PubMed1991

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.

Adenosine Triphosphate↗
PubMed1991

Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA.

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).

Autoantigens↗
PubMed1991

Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.

Activator 1 (A1) is a multiprotein complex which is essential for proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase delta (pol delta) activity and efficient in vitro DNA synthesis in the SV40 dipolymerase replication system. In this report, we describe the isolation of A1 from HeLa cytosolic extracts. A1 stimulated pol delta activity in singly primed phi X174 DNA or (dA)4500.oligo(dT)12-18 in reactions containing PCNA, single-stranded DNA binding protein (SSB), and ATP. Using this assay, A1 has been extensively purified. Purified preparations contained five discrete subunits of 145, 40, 38, 37, and 36.5 kDa. ATP hydrolysis to ADP and Pi is essential for A1-dependent pol delta activity, and we have shown that A1 contains an intrinsic ATPase which is stimulated by DNA. The DNA-dependent hydrolysis of ATP can be stimulated by PCNA and further activated by PCNA plus the human single-stranded DNA binding protein. These stimulatory effects were observed with (dA)4500.oligo(dT)12-18, but were not detected with each poly-deoxynucleotide alone. Furthermore, A1 formed a complex with (dA)4500.oligo(dT)12-18 which could be measured by nitrocellulose binding. No complex with (dA)4500 or oligo(dT)12-18 alone was detected by this procedure. Data are also presented which indicate that A1, in conjunction with PCNA, functions as a primer-recognition factor for pol delta, increasing its ability to utilize low levels of primer ends, but it does not increase the size of the DNA products. A1 also markedly reduced the amount of PCNA required for pol delta activity on a multiply primed DNA suggesting that PCNA interacts with A1 at the primer end. These multiple effects of A1 closely resemble the properties of the multisubunit protein RF-C described by Tsurimoto and Stillman (Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023-1027).

Adenosine Triphosphatases↗
PubMed1991

Differential induction of structural changes in the simian virus 40 origin of replication by T antigen.

The ATP-dependent binding of the simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication (ori) results in the structural distortion of two critical elements within flanking regions of ori and the untwisting of the DNA helix. We examined the effect of changes in temperature, ATP concentration, and other reaction parameters on the generation of these DNA structural changes. We found that induction of the two localized structural transitions were highly and differentially sensitive to reaction conditions. Significant distortion of the early palindrome element, shown previously to result from DNA melting, required low levels of ATP (10 to 30 microM) but temperatures above 25 degrees C. Distortion of the AT tract occurred at low temperatures (5 degrees C) but required relatively high concentrations of ATP (greater than 300 microM). Thus, T antigen can induce structural changes within one critical element of ori without generating significant structural distortion within the second element. The response of ori untwisting to reaction conditions generally increased in parallel with or fell intermediate between the inductions of localized structural transitions. We suggest that ori untwisting and localized structural distortions are interdependent consequences of T-antigen binding to ori. These results suggest a model for the structural events occurring during the initial steps of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed1991

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.

Antigens, Polyomavirus Transforming↗
PubMed1990

The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication.

Human single-stranded DNA binding protein (human SSB) is a multisubunit protein containing polypeptides of 70, 34, and 11 kDa that is required for SV40 DNA replication in vitro. In this report we identify the functions of the SSB and its individual subunits in SV40 DNA replication. The 70 kDa subunit was found to bind to single-stranded DNA, whereas the other subunits did not. Four monoclonal antibodies against human SSB were isolated which inhibited SV40 DNA replication in vitro. The antibodies have been designated alpha SSB70A, alpha SSB70B, alpha SSB70C, and alpha SSB34A to indicate which subunits are recognized. Immunolocalization experiments indicated that human SSB is a nuclear protein. Human SSB is required for the SV40 large tumor antigen-catalyzed unwinding of SV40 DNA and stimulates DNA polymerases (pol) alpha and delta. The DNA unwinding reaction and stimulation of pol delta were blocked by alpha SSB70C, whereas the stimulation of pol alpha by human SSB was unaffected by this antibody. Conversely, alpha SSB70A, -70B, and -34A inhibited the stimulation of pol alpha, but they had no effect on DNA unwinding and pol delta stimulation. None of the antibodies inhibited the binding of SSB to single-stranded DNA. These results suggest that DNA unwinding and stimulation of pol alpha and pol delta are required functions of human SSB in SV40 DNA replication. The human SSB 70-kDa subunit appears to be required for DNA unwinding and pol delta stimulation, whereas both the 70- and 34-kDa subunits may be involved in the stimulation of pol alpha.

Antibodies, Monoclonal↗
PubMed1990

Assemblage of the prespliceosome complex with separated fractions isolated from HeLa cells.

The first ATP-dependent complex formed in pre-mRNA splicing is the prespliceosome, a 30 S complex. This reaction was investigated using partially purified fractions isolated from nuclear extracts of HeLa cells. Previous studies (Furneaux, H. M., Perkins, K. K., Freyer, G. A., Arenas, J., and Hurwitz, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4351-4355) have shown that DEAE-cellulose chromatography of nuclear extracts yielded two fractions (fractions I and II, eluted at 0.2 and 1 M NaCl, respectively) which carried out pre-mRNA splicing only when combined. Fraction II, alone and in the presence of ATP, supported the formation of the 30 S complex. In this report, we have separated fraction II into ribonucleoprotein and protein-rich fractions by isopycnic banding in CsCl. The combination of these two fractions completely replaced fraction II in prespliceosome formation; when supplemented with fraction Ib (1 M NaCl Biorex fraction derived from fraction I), the preparations supported spliceosome formation; when supplemented with fraction I, they yielded spliced products. The CsCl fractions, like fraction II, efficiently converted pre-mRNA to the 30 S complex with high yields (30-70%). The 30 S complex was shown to contain pre-mRNA complexed to U2 small ribonucleoproteins and small amounts of U1 small ribonucleoproteins. The 30 S complex protected a 50-nucleotide region at the 3'-end of the intron from T1 RNase attack. This region included sequences spanning the branch site, the polypyrimidine stretch and the AG dinucleotide of the 3'-splice site. When the 30 S complex was first generated with partially purified fractions, followed by the addition of a large amount of poly(U) or unlabeled pre-mRNA, the 30 S complex could be chased into a 55 S spliceosome complex by the addition of fraction Ib. These results support the conclusion, initially derived from kinetic data, that the 30 S complex is a precursor of the 55 S complex.

Base Sequence↗
PubMed1990

Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1.

In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase delta (pol delta) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA). We have examined the interaction of these proteins with (dA)4500.(dT)12-18 by measuring their ability to form stable complexes with this DNA. In the presence of ATP, A1, PCNA, and pol delta formed a stable complex with DNA that could be isolated by gel filtration. Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT). While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA. The temporal requirements for complex formation were determined. A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.DNA complex, while pol delta was added last. Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability. The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5'-[gamma-thio]triphosphate as well as ATP. However, the binding of pol delta to the PCNA.A1-DNA complex was observed only when the latter complex was formed in the presence of ATP. The complete complex was formed after incubation at 37 degrees C for 2 min, whereas no complex was detected after incubation at 0 degree C. These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.

Adenosine Triphosphate↗