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J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 127 records · Page 7Linked to original sources
PubMed1990

Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.

The synthesis of oligoribonucleotides by DNA primase in the presence of duplex DNA containing the simian virus 40 (SV40) origin of replication was examined. Small RNA chains (10-15 nucleotides) were synthesized in the presence of the four common ribonucleoside triphosphates, SV40 large tumor antigen (T antigen), the human DNA polymerase alpha (pol alpha)-DNA primase complex, the human single-stranded DNA-binding protein (HSSB), and topoisomerase I isolated from HeLa cells. The DNA primase-catalyzed reaction showed an absolute requirement for T antigen, HSSB, and pol alpha. The requirement for HSSB was not satisfied by other SSBs that can support the T-antigen-catalyzed unwinding of DNA containing the SV40 origin of replication. Oligoribonucleotide synthesis occurred with a lag that paralleled the lag observed in DNA synthesis. These results indicate that the specificity for the HSSB in the SV40 replication reaction is due to the pol alpha-primase-mediated synthesis of the Okazaki fragments. In contrast to this specificity, the elongation of Okazaki fragments can be catalyzed by a variety of different DNA polymerases, including high levels of pol alpha, the polymerase delta holoenzyme, T4 polymerase holoenzyme, the Escherichia coli polymerase III holoenzyme, and other polymerases. These observations suggest that leading-strand synthesis in the in vitro SV40 replication system can be nonspecific.

DNA Polymerase II↗
PubMed1989

Alterations in the polypyrimidine sequence affect the in vitro splicing reactions catalyzed by HeLa cell-free preparations.

The polypyrimidine tract, located at the 3' end of intron 1 of the adenovirus major late transcript, was studied for its role in splicing using cell-free preparations isolated from HeLa cells. A plasmid (pIz) was constructed in which seven purine bases were substituted for pyrimidine bases within the 14-nucleotide polypyrimidine sequence. Runoff transcripts extending to the middle of intron 2 were tested for their ability to support in vitro splicing. The efficiency of these reactions was compared with pre-mRNA transcripts made from the wild-type nonmutated plasmid (p1-2). Neither spliced products nor splicing intermediates were detected in reactions with the pIz pre-mRNA. The formation of the nucleoprotein complexes involved in splicing was examined with this altered pre-mRNA. No 55 S splicing complex was detected and only low levels of the 30 S presplicing complex formed (30-fold less than with wild-type pre-mRNA). However, when a longer runoff transcript was prepared from the polypyrimidine mutated plasmid pIz, spliced RNA was formed. This activity required specific downstream sequences, since transcripts produced from pIz which contained substituted downstream sequences were not spliced. Although intron 2 of the adenovirus major late transcript does not contain a discernible 3' polypyrimidine sequence, pre-mRNA (p2-3) containing this intron was efficiently spliced. However, when the 3' region of intron 2 was substituted for the polypyrimidine sequence of intron 1, the resulting pre-mRNA did not support efficient splicing in vitro. However, when the polypyrimidine sequence of intron 1 was substituted for the sequence at the 3' end of intron 2, efficient splicing occurred, and the rate of formation of splicing intermediates and the accumulation of nucleoprotein complexes was greater than with the wild-type pre-mRNA (p2-3).

Adenoviruses, Human↗
PubMed1989

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication.

Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.

Adenosine Triphosphate↗
PubMed1989

Initiation of simian virus 40 DNA replication in vitro: pulse-chase experiments identify the first labeled species as topologically unwound.

A distinct unwound form of DNA containing the simian virus 40 (SV40) origin is produced in replication reactions carried out in mixtures containing crude fractions prepared from HeLa cells. This species, termed form UR, comigrates on chloroquine-containing agarose gels with the upper part of the previously described heterogeneous highly unwound circular DNA, form U. As with form U, formation of form UR is dependent upon the SV40 tumor (T) antigen. Pulse-chase experiments demonstrate that the first species to incorporate labeled deoxyribonucleotides comigrates with form UR. Restriction analyses of the products of the pulse-chase experiments show that initiation occurs at the SV40 origin and then proceeds outward in a bidirectional manner. These experiments establish form UR as the earliest detectable substrate for SV40 DNA replication and suggest that SV40 DNA replication initiates on an unwound species.

Animals↗
PubMed1989

Studies on the DNA elongation inhibitor and its proliferating cell nuclear antigen-dependent control in simian virus 40 DNA replication in vitro.

A 120-kDa protein that blocks DNA termini has been purified from extracts of HeLa cells. This protein inhibits the action of a number of enzymes that catalyze reactions involving the 5' and 3' ends of DNA (DNA ligase, 3' and 5' exonucleases, and DNA polymerase alpha). The 120-kDa protein blocks the synthesis of long DNA chains that are normally formed during simian virus 40 DNA replication, causing the accumulation of small DNA fragments. The effects of this protein can be reversed by the addition of proliferating cell nuclear antigen and other protein fractions (activators).

Antigens, Neoplasm↗
PubMed1989

Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.

Antigens, Neoplasm↗
PubMed1989

Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta.

The human single-stranded-DNA binding protein (human SSB) is required for simian virus 40 (SV40) DNA replication in vitro. SV40 large tumor antigen and human SSB can support extensive unwinding of SV40 origin-containing DNA in the presence of ATP and a topoisomerase that relieves positive superhelicity. Although SSBs from viral and prokaryotic sources substituted for human SSB in the DNA-unwinding reaction, they did not substitute in the replication of SV40 DNA. The specificity for human SSB in SV40 DNA replication can be explained, at least in part, by the finding that DNA polymerase alpha was stimulated 10-fold by human SSB but not by other SSBs. Human SSB also stimulated proliferating-cell nuclear antigen-dependent DNA polymerase delta; however, other SSBs stimulated this polymerase as well.

DNA Polymerase II↗
PubMed1989

Development of an effective short introductory course in diagnostic radiology.

As part of a general medical curriculum renewal, a new one-week course in diagnostic radiology was given to students at the beginning of their third year. The course format consisted of didactic presentations to the entire class, a single afternoon session of small group discussions of unknowns, and three daily case-of-the-day unknowns with prizes for correct responses. One hundred fifty-eight students were tested on the first and last days of the course; testing consisted of two parallel 46-item multiple choice questions given in a crossed design. Questions covered radiographic anatomy and pathology as well as proper test ordering. Differences in achievement before and after the course as measured by the pre- and post-tests were highly significant. The class achieved a mean of 27 (59%) correct responses on the pre-test and a mean of 37 (80%) on the post-test (F(1156) = 15.79; P less than .001). The reliability coefficient of the pre- and post-tests was about .30. A course critique was returned by 88 students. There was anticipated variation in the evaluations of the different didactic lecturers. Instructors receiving the highest scores were cited for their enthusiasm, clarity, and the relevance of their presentation. Conversely, those receiving low marks were cited as being unenthusiastic and boring, merely presenting example after example of radiographic abnormalities with little explanation. The afternoon small group discussions were perceived as the best part of the course. Despite the fact that moderator experience ranged from second year resident to full professor, the seminars received uniformly positive comments.

Education, Medical↗
PubMed1988

Complete enzymatic synthesis of DNA containing the SV40 origin of replication.

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.

Antigens, Polyomavirus Transforming↗
PubMed1988

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin.

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.

Bacteriophage phi X 174↗
PubMed1988

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. II. Effects of DNA replication of mutations in the 30-nucleotide icosahedral bacteriophage origin.

phi X174 viral strand circular DNA can be synthesized in vitro from phi X174 replicative form I (RFI) DNA in the presence of the phi X A protein, the Escherichia coli DNA polymerase III elongation system, the E. coli rep helicase, and the E. coli single-stranded DNA binding protein. M13mp9 or pBR322 RFI DNAs containing a 30-base pair sequence found at the phi X origin of replication supported phi X A protein synthesis as well as the phi X template, giving rise to a net molar excess of deoxynucleotide incorporation. In this paper, we show that mutations in positions 1-3 of the 30-nucleotide origin replicated at a lower efficiency than plasmids containing the wild-type origin, because of a deficiency in the reinitiation reaction. Mutations in positions 4-7, upstream of the phi X A protein cleavage site, failed to support replication because of their inability to support nicking. An origin containing a mutation at the residue to which the phi X A protein is covalently linked to the DNA was an active template that supported a net molar excess of incorporation. Mutations at the 3' end of the origin region, retaining only the first 21-25 nucleotides of the 30-base pair origin, failed to support replication because of impaired binding of the phi X A protein to the template and consequently poor nicking. A construct bearing the first 28 nucleotides of the origin supported wild-type replication, as did a plasmid containing a 28-mer origin with a point mutation at position 26, but this latter construct also appeared to be partially deficient in phi X A protein binding activity. These results are consistent with the presence of a phi X A protein binding domain at the 3' end of the origin.

Bacteriophage phi X 174↗
PubMed1988

Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis. III. Replication of DNA containing two viral replication origins.

Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.

Bacteriophage phi X 174↗
PubMed1988

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway.

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.

Bacteriophage phi X 174↗
PubMed1988

Initiation of adenovirus DNA replication. I. Mechanism of action of a host protein required for replication of adenovirus DNA templates devoid of the terminal protein.

The in vitro replication of adenovirus (Ad) DNA covalently attached to the 55-kDa terminal protein requires at least five proteins including the 80-kDa preterminal protein, the Ad DNA polymerase, the Ad DNA binding protein, nuclear factor I, and topoisomerase I. The replication of Ad DNA templates devoid of the terminal protein requires an additional protein, designated factor pL, which has been purified from uninfected HeLa cell nuclei (Guggenheimer, R. A., Nagata, K., Kenny, M., and Hurwitz, J. (1984) J. Biol. Chem. 259, 7815-7825). Factor pL has been found to contain an intrinsic 5'----3' exonuclease activity. When Ad DNA templates lacking the terminal protein were pretreated with factor pL, the requirement for factor pL in the replication reaction was abolished. Synthetic partially duplex oligonucleotide templates containing Ad origin sequences were constructed in order to determine the structure of the DNA molecules that are active in the absence of factor pL. These experiments indicated that factor pL degrades the 5'-end of the nontemplate (displaced) strand of the Ad origin thereby creating a single-stranded region at the 3'-end of the template strand. Such DNAs are competent for initiation of Ad DNA replication in the absence of factor pL but remain dependent on nuclear factor 1.

Adenoviridae↗
PubMed1988

Initiation of adenovirus DNA replication. II. Structural requirements using synthetic oligonucleotide adenovirus templates.

Adenovirus (Ad) virions contain a 55-kDa terminal protein covalently linked to both 5'-ends of the linear duplex DNA genome. The origin of DNA replication is contained within the terminal 50 base pair of the inverted terminal repeats. In the accompanying paper (Kenny, M. K., Balogh, L. A., and Hurwitz, J. (1988) J. Biol. Chem. 263, 9801-9808), it was demonstrated that synthetic oligonucleotide templates which contain the Ad origin, but lack the 55-kDa terminal protein, can serve as templates for the initiation of Ad DNA replication. Partially duplex oligonucleotides that lacked up to 14 nucleotides from the 5'-end of the nontemplate (displaced) strand supported initiation as much as 20-fold more efficiently than fully duplex oligonucleotides. The removal of 18 nucleotides or more from the 5'-end of the displaced strand resulted in a sharp decrease in the ability of the DNA templates to support initiation. The poor template efficiency of certain DNAs could be explained by their inability to bind nuclear factor I. The initiation efficiency observed with other DNAs correlated with their ability to bind the preterminal protein-Ad DNA polymerase complex. At low concentrations of the Ad DNA-binding protein, protein-primed initiation was also observed on single-stranded DNAs. The single-stranded template strand of the Ad origin was at least 5-20-fold better at supporting initiation than other single-stranded DNAs. These findings suggest a model in which the 3'-end of the template strand is rendered single-stranded as a prerequisite for initiation of Ad DNA replication.

Adenosine Triphosphate↗
PubMed1988

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity.

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.

Antigens, Polyomavirus Transforming↗
PubMed1988

Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.

Replication of simian virus 40 (SV40) DNA is dependent upon the binding of the viral T-antigen to the SV40 origin of replication. Structural changes in the origin of replication induced by binding of T-antigen were probed by chemical modifications of the DNA. In the presence of ATP, T-antigen rendered two of three domains in the SV40 core origin hypersensitive to attack by either dimethyl sulfate or potassium permanganate (KMnO4). One of these domains, the early palindrome, was shown to contain an 8-bp region of melted DNA as determined from methylation of cytosine residues and by nuclease S1 cleavage of methylated DNA. DNA melting was not dependent upon either the hydrolysis of ATP or the binding of T-antigen to an adjacent site (site I). A second domain, the A/T element, was extensively modified by KMnO4 but no significant melting was detected. Rather, the pattern of modification indicates that T-antigen caused a conformational change of the double-stranded DNA in this region. These results suggest that T-antigen, in the presence of ATP, destabilizes the SV40 origin by melting and structurally deforming two flanking regions within the core origin sequence. These DNA structural changes may provide access to other replication factors, allowing complete denaturation of the SV40 origin and the initiation of SV40 DNA replication.

Adenosine Triphosphate↗