PubMed Health⌕ Search

Biomedical subjects

J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Reconstitution of proliferating cell nuclear antigen-dependent repair of apurinic/apyrimidinic sites with purified human proteins.

An apurinic/apyrimidinic (AP) site is one of the most abundant lesions spontaneously generated in living cells and is also a reaction intermediate in base excision repair. In higher eukaryotes, there are two alternative pathways for base excision repair: a DNA polymerase beta-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Here we have reconstituted PCNA-dependent repair of AP sites with six purified human proteins: AP endonuclease, replication factor C, PCNA, flap endonuclease 1 (FEN1), DNA polymerase delta, and DNA ligase I. The length of nucleotides replaced during the repair reaction (patch size) was predominantly two nucleotides, although longer patches of up to seven nucleotides could be detected. Neither replication protein A nor Ku70/80 enhanced the repair activity in this system. Disruption of the PCNA-binding site of either FEN1 or DNA ligase I significantly reduced efficiency of AP site repair but did not affect repair patch size.

Base Sequence↗
PubMed1999

Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.

Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase alpha-primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase delta assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single-stranded DNA-binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition-based protein switches in which two proteins vie for contact with RPA.

Binding, Competitive↗
PubMed1999

Identification and reconstitution of the origin recognition complex from Schizosaccharomyces pombe.

The origin recognition complex (ORC), first identified in Saccharomyces cerevisiae (sc), is a six-subunit protein complex that binds to DNA origins. Here, we report the identification and cloning of cDNAs encoding the six subunits of the ORC of Schizosaccharomyces pombe (sp). Sequence analyses revealed that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts from S. cerevisiae, Xenopus, Drosophila, and human. In contrast, both spOrc3 and spOrc6 subunits are poorly conserved. As reported by Chuang and Kelly [(1999) Proc. Natl. Acad. Sci. USA 96, 2656-2661], the C-terminal region of spOrc4 is also conserved whereas the N terminus uniquely contains repeats of a sequence that binds strongly to AT-rich DNA regions. Consistent with this, extraction of S. pombe chromatin with 1 M NaCl, or after DNase I treatment, yielded the six-subunit ORC, whereas extraction with 0.3 M resulted in five-subunit ORC lacking spOrc4p. The spORC can be reconstituted in vitro with all six recombinant subunits expressed in the rabbit reticulocyte system. The association of spOrc4p with the other subunits required the removal of DNA from reaction mixture by DNase I. This suggests that a strong interaction between spOrc4p and DNA can prevent the isolation of the six-subunit ORC. The unique DNA-binding properties of the spORC may contribute to our understanding of the sequence-specific recognition required for the initiation of DNA replication in S. pombe.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum deltaH.

We describe here the isolation and characterization of a B-type DNA polymerase (PolB) from the archaeon Methanobacterium thermoautotrophicum DeltaH. Uniquely, the catalytic domains of M. thermoautotrophicum PolB are encoded from two different genes, a feature that has not been observed as yet in other polymerases. The two genes were cloned, and the proteins were overexpressed in Escherichia coli and purified individually and as a complex. We demonstrate that both polypeptides are needed to form the active polymerase. Similar to other polymerases constituting the B-type family, PolB possesses both polymerase and 3'-5' exonuclease activities. We found that a homolog of replication protein A from M. thermoautotrophicum inhibits the PolB activity. The inhibition of DNA synthesis by replication protein A from M. thermoautotrophicum can be relieved by the addition of M. thermoautotrophicum homologs of replication factor C and proliferating cell nuclear antigen. The possible roles of PolB in M. thermoautotrophicum replication are discussed.

Amino Acid Sequence↗
PubMed1999

The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity.

Proliferating cell nuclear antigen (PCNA), the processivity factor (sliding clamp) of DNA polymerases (Pols), plays essential roles in DNA metabolism. In this report, we examined the functional role of the C-terminal region of Schizosaccaromyces pombe PCNA both in vitro and in vivo. The deletion or Ala substitution of the last 9 aa (252-260A), as well as Ala replacement of only 4 aa (252-255A) at the C terminus, failed to substitute for the wild-type PCNA protein for cell growth in S. pombe. Two other PCNA mutant proteins, A251V and K253E, exhibited cold-sensitive phenotypes. Several yeast strains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage. The ability of the mutant PCNA proteins to stimulate DNA synthesis by Poldelta and Polepsilon also was studied in vitro. The mutant proteins that did not support cell growth and a mutant protein containing a single amino acid substitution at position 252, where Pro is replaced by Ala, stimulated Poldelta and Polepsilon activities poorly. All mutant PCNA proteins, however, were assembled around DNA by the clamp loader, replication factor C, efficiently. Thus, the C-terminal region of PCNA is important for interactions with both Poldelta and Polepsilon and for cell survival after DNA damage. The C terminus of sliding clamps from other organisms has been shown to be important for clamp loading as well as polymerase interactions. The relationship between the conserved sequence in this region in different organisms is discussed.

Amino Acid Sequence↗
PubMed1999

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or epsilon)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

Amino Acid Substitution↗
PubMed1999

Androgens in preeclampsia.

OBJECTIVE: The purpose of the study was to determine whether maternal serum levels of androgens, especially testosterone, are higher in patients with preeclampsia than in matched normotensive control subjects. STUDY DESIGN: Serum testosterone, dehydroepiandrosterone sulfate, sex hormone binding globulin, and estradiol levels were measured in 16 subjects in the third trimester of pregnancy with documented preeclampsia and 26 healthy, normotensive women with similar maternal and gestational ages. All subjects were primigravid women with singleton pregnancies who were seen in the labor and delivery department at North Oakland Medical Centers in Pontiac, Mich. RESULTS: Total testosterone and free testosterone levels were significantly higher in patients with preeclampsia (213.6 +/- 25.9 ng/dL and 0.5 +/- 0.1 ng/dL, respectively) than in the control group (154.5 +/- 14.8 ng/dL and 0. 3 +/- 0.03 ng/dL, respectively). There were no significant differences in sex hormone binding globulin, dehydroepiandrosterone sulfate, and estradiol concentrations. There were also no significant differences in maternal age, gestational age, body mass index, and neonatal sex. CONCLUSION: Levels of the potent androgen testosterone were significantly higher in primigravid women with preeclampsia than in normotensive women with similar gestational and maternal ages. This difference may indicate a role for testosterone in the pathogenesis of preeclampsia.

Adult↗
PubMed1999

The human RNA helicase A (DDX9) gene maps to the prostate cancer susceptibility locus at chromosome band 1q25 and its pseudogene (DDX9P) to 13q22, respectively.

RNA helicase A is the homolog of the Drosophila maleless protein, an essential factor involved in dosage compensation, and plays a crucial role in early development in mammals. Here, we have mapped the human RNA helicase A (DDX9) gene to the major susceptibility locus for prostate cancer at chromosome band 1q25, and its pseudogene (DDX9P) to the band 13q22 by fluorescence in situ hybridization, somatic cell hybrid analysis, and assignment of YAC clones, respectively.

Amino Acid Sequence↗
PubMed1998

RNA helicase A is essential for normal gastrulation.

RNA helicase A (RHA) is the human homologue of the Drosophila maleless protein, an essential factor for the development of male flies. Recently, it was shown that RHA cooperates with the cAMP-responsive element in mediating the cAMP-dependent transcriptional activation of a number of genes. Due to the participation of cAMP as a second messenger in a number of signaling pathways, we examined the function of RHA during mammalian embryogenesis. To examine the role(s) of RHA in mammalian development, RHA knockout mice were generated by homologous recombination. Homozygosity for the mutant RHA allele led to early embryonic lethality. Histological analysis, combined with terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) reactions of RHA-null embryos, revealed marked apoptotic cell death specifically in embryonic ectodermal cells during gastrulation. RNA in situ analyses of the expression of HNF-3beta and Brachyury, two molecular markers for gastrulation, showed that RHA-null embryos at days 7.5 and 8.5 expressed both HNF-3beta and Brachyury in a pattern similar to those of pre- and early streak stages of embryos, respectively. These observations indicate that RHA is necessary for early embryonic development and suggest the requirement of RHA for the survival and differentiation of embryonic ectoderm.

Animals↗
PubMed1998

ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits.

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.

Adenosine Triphosphate↗
PubMed1998

Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57KIP2 requires binding to proliferating cell nuclear antigen.

Proper control of the mammalian cell cycle requires the function of cyclin-dependent kinase (CDK) inhibitors. The p21 family currently includes three distinct genes, p21, p27(Kip1), and p57(Kip2), that share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin complexes. The p21 protein also binds to proliferating cell nuclear antigen (PCNA) through a separate C-terminal domain affecting DNA replication and repair. The p27 and p57 proteins also each contain unique C-terminal domains whose functions are unknown. Here we show that the human p57 protein, like p21, contains a PCNA-binding domain within its C terminus that, when separated from its N-terminal CDK-cyclin binding domain, can prevent DNA replication in vitro and S phase entry in vivo. Disruption of either CDK/cyclin or PCNA binding partially reduced p57's ability to suppress myc/RAS-mediated transformation in primary cells, while loss of both inhibitory functions completely eliminated p57's suppressive activity. Thus, control of cell cycle and suppression of cell transformation by p57 require both CDK and PCNA inhibitory activity, and disruption of either or both functions may lead to uncontrolled cell growth.

Amino Acid Sequence↗
PubMed1998

Processivity of DNA polymerases: two mechanisms, one goal.

Replicative DNA polymerases are highly processive enzymes that polymerize thousands of nucleotides without dissociating from the DNA template. The recently determined structure of the Escherichia coli bacteriophage T7 DNA polymerase suggests a unique mechanism that underlies processivity, and this mechanism may generalize to other replicative polymerases.

Bacteriophage T4↗
PubMed1998

Molecular analysis of the cDNA and genomic DNA encoding mouse RNA helicase A.

RNA helicase A is an enzyme that possesses both RNA and DNA helicase activities. In this report, we describe the isolation of a mouse cDNA encoding RNA helicase A. The deduced amino acid sequence derived from mouse RNA helicase A cDNA exhibits 87 and 47% identity to its human and Drosophila homologs, respectively. Using Southern blot analysis employing a mouse backcross panel, we have assigned the mouse RNA helicase A gene to chromosome 1, mapping near the D1Bir20 locus at MGD position 67. Northern blot and primer extension analyses indicate that, although its level is variable, RNA helicase A appears to be expressed from a single transcription start site in all tissues tested. Sequence analysis of the upstream genomic DNA revealed that the promoter region lacks a TATA box and contains two high-affinity sites for Sp1, one ISRE, a binding site for interferon regulatory factor, and three AP2-binding sites. These findings suggest that the transcriptional regulation of the RNA helicase A gene is complex.

Amino Acid Sequence↗
PubMed1998

Human RPA (hSSB) interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus.

RPA is the replicative single-strand DNA (ssDNA) binding protein of eukaryotic chromosomes. This report shows that human RPA interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus (EBV). RPA binds to EBNA1 both in solution, and when EBNA1 is bound to the EBV origin. RPA is a heterotrimer, and the main contact with EBNA1 is formed through the 70 kDa subunit of RPA, the subunit which binds to ssDNA. We propose that this interaction between RPA and EBNA1 is an early step in activation of the latent origin of EBV.

DNA, Viral↗
PubMed1998

Protein-PCNA interactions: a DNA-scanning mechanism?

Proliferating-cell nuclear antigen (PCNA) plays an essential role in nucleic-acid metabolism in all eukaryotes. The PCNA protein interacts with a large number of proteins. These proteins can be divided into two groups: the first contains proteins that have a known enzymatic activity; the second contains regulatory proteins that are involved in cell-cycle progression, checkpoint control and cellular differentiation. Interestingly, all of the enzymes known to interact with PCNA either recognize specific structures on DNA or have limited DNA-sequence specificity. Proteins that have low sequence specificities could utilize PCNA as an adapter in order to interact with their DNA substrates.

Animals↗
PubMed1998

Isolation, characterization, and molecular cloning of a protein (Abp2) that binds to a Schizosaccharomyces pombe origin of replication (ars3002).

The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S. pombe ARS consensus sequence previously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203-2209, 1988). Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abpl [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502-507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.

Amino Acid Sequence↗