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J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 55 records · Page 3Linked to original sources
PubMed1997

Isolation of human and fission yeast homologues of the budding yeast origin recognition complex subunit ORC5: human homologue (ORC5L) maps to 7q22.

Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S. pombe, and S. cerevisiae Orc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified the S. pombe ORC5 gene and located it in a cosmid mapped on chromosome II.

Amino Acid Sequence↗
PubMed1997

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

Apoptosis↗
PubMed1997

DNA polymerase delta isolated from Schizosaccharomyces pombe contains five subunits.

DNA polymerase delta (pol delta) plays an essential role in DNA replication, repair, and recombination. We have purified pol delta from Schizosaccharomyces pombe more than 10(3)-fold and demonstrated that the polymerase activity of purified S. pombe pol delta is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol delta complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.

Amino Acid Sequence↗
PubMed1997

RNA helicase A mediates association of CBP with RNA polymerase II.

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Here we show that complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor mle. In transient transfection assays, RHA was found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. As a mutation in RHA that compromised its helicase activity correspondingly reduced CREB-dependent transcription, we propose that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters.

Animals↗
PubMed1997

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe.

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Amino Acid Sequence↗
PubMed1997

A complex consisting of human replication factor C p40, p37, and p36 subunits is a DNA-dependent ATPase and an intermediate in the assembly of the holoenzyme.

Human replication factor C (hRFC) is a multi-subunit protein complex capable of supporting proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis by DNA polymerases delta and epsilon. The hRFC complex consists of five different subunits with apparent molecular masses of 140, 40, 38, 37, and 36 kDa. We have previously reported the expression of a three-subunit core complex, consisting of the p40, p37, and p36 subunits following coupled in vitro transcription-translation of the cDNAs encoding these proteins (Uhlmann, F., Cai, J., Flores-Rozas, H., Dean, F. B., Finkelstein, J. , O'Donnell, M., and Hurwitz, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6521-6526). Here we describe the isolation of a stable complex composed of the p40, p37, and p36 subunits of hRFC from baculovirus-infected insect cells. The purified p40.p37.p36 complex, like the five-subunit RFC, contained DNA-dependent ATPase activity that was stimulated by PCNA, preferentially bound to primed DNA templates, interacted with PCNA, and was capable of unloading PCNA from singly-nicked circular DNA. In contrast to the five-subunit RFC, the three-subunit core complex did not load PCNA onto DNA. The p40. p37.p36 complex inhibited the elongation of primed DNA templates catalyzed by the DNA polymerase delta holoenzyme. Incubation of the p40.p37.p36 complex with the hRFC p140 and p38 subunits formed the five-subunit hRFC complex that supported PCNA-dependent DNA synthesis by DNA polymerase delta.

Adenosine Triphosphatases↗
PubMed1997

The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation.

Drosophila maleless (mle) is required for X chromosome dosage compensation and is essential for male viability. Maleless protein (MLE) is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II. In this report, we demonstrate that MLE protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. Using site-directed mutagenesis, we created a mutant of MLE (mle-GET) that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis showed that this mutation abolished both NTPase and helicase activities of MLE but affected the ability of MLE to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein could still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, MSL-1, but failed to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of MLE for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes.

Acid Anhydride Hydrolases↗
PubMed1997

Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro.

Human single-stranded DNA-binding protein (HSSB, also called RPA), is a heterotrimeric complex that consists of three subunits, p70, p34, and p11. HSSB is essential for the in vitro replication of SV40 DNA and nucleotide excision repair. It also has important functions in other DNA transactions, including DNA recombination, transcription, and double-stranded DNA break repair. The p34 subunit of HSSB is phosphorylated in a cell cycle-dependent manner. Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro. In this study, we show that efficient phosphorylation of HSSB-p34 by DNA-PK requires Ku as well as DNA. The DNA-PK phosphorylation sites in HSSB-p34 have been mapped at Thr-21 and Ser-33. Kinetic studies demonstrated that a phosphate residue is first incorporated at Thr-21 followed by the incorporation of a second phosphate residue at Ser-33. We also identified Ser-29 as the major Cdc2 kinase phosphorylation site in the p34 subunit.

Amino Acid Sequence↗
PubMed1997

Deletion analysis of the large subunit p140 in human replication factor C reveals regions required for complex formation and replication activities.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases delta and epsilon. RFC contains multiple activities, including its ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template allowing processive DNA chain elongation. Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36), and RFC activity can be reconstituted from the five cloned gene products. To characterize the role of the large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required for complex formation with the four small subunits. Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of an RFC complex with enhanced activity in replication and PCNA loading. Deletion of additional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupted RFC replication function. DNA primer end recognition and PCNA binding activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished.

DNA↗
PubMed1997

Identification of regions within the four small subunits of human replication factor C required for complex formation and DNA replication.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases delta and epsilon. RFC binds to a DNA primer end and loads PCNA onto DNA in an ATP-dependent reaction. The five RFC subunits p140, p40, p38, p37, and p36, all of which are required to form the active RFC complex, share regions of high homology including the defined RFC boxes II-VIII. RFC boxes III and V constitute a putative ATP binding site, whereas the function of the other conserved boxes is unknown. To study the individual subunits in the RFC complex and the role of the RFC boxes, deletion mutations were created in all subunits. Sequences close to the C terminus of each of the small subunits are required for formation of the five subunit complex. A N-terminal region of the small subunits, containing the RFC homology box II, plays a critical role in the function of these subunits, deletion of which reduces but does not abolish RFC activity in loading PCNA onto DNA and in supporting an RFC-dependent replication reaction. The N termini of p37 and p40, although highly homologous, are not interchangeable, suggesting unique functions for the individual subunits.

DNA Repair↗
PubMed1997

The influence of the proliferating cell nuclear antigen-interacting domain of p21(CIP1) on DNA synthesis catalyzed by the human and Saccharomyces cerevisiae polymerase delta holoenzymes.

In eukaryotes, processive DNA synthesis catalyzed by DNA polymerases delta and epsilon (pol delta and epsilon) requires the proliferating cell nuclear antigen (PCNA). It has recently been shown that in humans (h), the PCNA function, required for both DNA replication and nucleotide excision repair, can be inactivated by p21(CIP1) due to a specific interaction between hPCNA and the carboxyl terminus of p21(CIP1). In this report, we show that Saccharomyces cerevisiae (S. cerevisiae) PCNA-dependent pol delta-catalyzed DNA synthesis was inhibited less efficiently than the human system by the intact p21(CIP1) protein and was unaffected by the p21(CIP1) carboxyl-terminal peptide (codons 139-160). This species-specific response of PCNA to p21(CIP1)-mediated inhibition of DNA synthesis results from a marked difference in the ability of h and S. cerevisiae PCNA to interact with p21(CIP1). As shown by binding studies using the surface plasmon resonance technique, hPCNA binds both full-length p21(CIP1) and the p21(CIP1) peptide-(139-160) stoichiometrically with a similar affinity (KD approximately 2.5 nM) while S. cerevisiae PCNA binds p21(CIP1) with approximately 10-fold less affinity and does not interact with the p21(CIP1) peptide-(139-160).

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1996

Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells.

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase delta or epsilon. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase delta-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.

Adenosine Triphosphatases↗
PubMed1996

Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA.

The crystal structure of the human DNA polymerase delta processivity factor PCNA (proliferating cell nuclear antigen) complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21(WAF1/CIP1) has been determined at 2.6 angstrom resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a beta sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21-PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly.

Amino Acid Sequence↗
PubMed1996

Studies on the in vitro phosphorylation of HSSB-p34 and -p107 by cyclin-dependent kinases. Cyclin-substrate interactions dictate the efficiency of phosphorylation.

Cyclin-dependent kinases (Cdks) are required for cell cycle progression. Two potentially significant Cdk substrates in human cells are the human single-stranded binding protein (HSSB or RPA), which plays an essential role in DNA replication, repair, and recombination, and the tumor suppressor p107 which acts to negatively regulate cell growth. In this report we describe the in vitro phosphorylation of these two proteins by Cdks in an attempt to understand how cyclin-substrate interactions direct phosphorylation efficiencies. We show that cyclin A-Cdk2 efficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or as a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 that is active in phosphorylating histone H1, does not support the phosphorylation of the p34 subunit of HSSB. We provide evidence that this differential phosphorylation results from a specific interaction between HSSB-p34 and cyclin A, but not cyclin E. Thus the observed cell cycle-dependent phosphorylation of HSSB-p34 at the G1 to S transition is most likely catalyzed by cyclin A-Cdk2 initiated by the direct interaction between cyclin A and the HSSB-p34 subunit. These studies are consistent with our previous observation that p107, which directly binds cyclin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B-associated kinases. Here we further demonstrate that cyclin A only complexes with p107 in its unphosphorylated form. These data suggest a catalytic mechanism by which Cdk acts: substrate targeting by a cyclin-substrate interaction followed by dissociation of the Cdk upon phosphate incorporation allowing the Cdk to become available for the next cycle of phosphorylation.

Base Sequence↗
PubMed1996

Transcription-positive cofactor 4 forms complexes with HSSB (RPA) on single-stranded DNA and influences HSSB-dependent enzymatic synthesis of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA in vitro requires a trimeric single-stranded DNA (ssDNA)-binding protein called HSSB or RPA. HSSB supports the unwinding of DNA containing the SV40 origin in the presence of the viral-encoded T antigen and is required for the initiation of RNA primer synthesis as well as processive elongation of DNA catalyzed by the DNA polymerase delta holoenzyme. In this report we show that the transcription positive cofactor 4 (PC4), a ssDNA-binding protein, forms complexes with HSSB on ssDNA and markedly affects the replication functions of HSSB. PC4 supports T antigen-catalyzed unwinding of SV40 origins in lieu of HSSB but inhibits both RNA primer synthesis and polymerase delta-catalyzed DNA chain elongation reactions. These inhibitory effects can be reversed by the addition of excess HSSB. Depending on the concentration of HSSB, PC4 is capable of either inhibiting or activating SV40 DNA replication measured in both mono- and dipolymerase systems. The possible role of PC4 in the initiation of DNA replication is discussed.

Antigens, Viral, Tumor↗
PubMed1996

Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe.

We have purified a single-stranded DNA-binding protein (SSB) from Schizosaccharomyces pombe (Sp) and have shown that it is composed of three subunits of 68, 30, and 12 kDa. The SpSSB supports T antigen-dependent unwinding of SV40 ori containing DNA, but is not functional in the SV40 in vitro replication reaction. All three genes that encode the SpSSB subunit have been isolated. The cloned cDNA of the ssb1(+), encoding the p68 subunit, contains 609 amino acids (68.3 kDa), while that of the ssb2(+), encoding the p30 subunit, contains a 279 amino acids (30.3 kDa). The genomic DNA clone of the p12 subunit gene (ssb3(+)) has 2 introns and an open reading frame of 104 amino acids (11.8 kDa). Significant homology is observed among the largest and middle subunits of eukaryotic SSBs, but there is poor homology among the smallest subunits. In addition, we have reconstituted the SpSSB complex by coexpression of all three subunits in Escherichia coli. The reconstituted complex is active in single-stranded DNA binding and the T antigen-dependent unwinding of SV40 ori DNA. Finally, we observed a cell cycle-dependent phosphorylation pattern of the p30 subunit of SpSSB, which is similar to that observed for the human and Saccharomyces cerevisiae SSB.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.

Animals↗