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Biomedical subjects

J Hurwitz

Publications and source records attributed to J Hurwitz.

At least 19 recordsLinked to original sources
PubMed2001

Targeting of human DNA polymerase iota to the replication machinery via interaction with PCNA.

Human DNA polymerase iota (hPoliota) promotes translesion synthesis by inserting nucleotides opposite highly distorting or noninstructional DNA lesions. Here, we provide evidence for the physical interaction of hPoliota with proliferating cell nuclear antigen (PCNA), and show that PCNA, together with replication factor C (RFC) and replication protein A (RPA), stimulates the DNA synthetic activity of hPoliota. In the presence of these protein factors, on undamaged DNA, the efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPoliota is increased approximately 80-150-fold, and this increase in efficiency results from a reduction in the apparent K(m) for the nucleotide. PCNA, RFC, and RPA also stimulate nucleotide incorporation opposite the 3'-T of the (6) thymine-thymine (T-T) photoproduct and opposite an abasic site. The interaction of hPoliota with PCNA implies that the targeting of this polymerase to the replication machinery stalled at a lesion site is achieved via this association.

Base Sequence↗
PubMed2001

The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein.

The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) autonomously replicating sequence 1 (ars1) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminal-deleted spOrc4 subunit (spORC(Delta N-Orc4)), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORC(Delta N-Orc4) and the Delta N-spOrc4p alone did not bind significantly to ars1. These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity. DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich sequence regions of ars1. These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the S. pombe ars1. These studies indicate that because of its unique Orc4 subunit, S. pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae.

AT Rich Sequence↗
PubMed2001

Purification and characterization of human DNA damage checkpoint Rad complexes.

Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

Adenosine Triphosphatases↗
PubMed2001

Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures.

The minichromosome maintenance (Mcm) proteins 2-7 are required for both the initiation and elongation steps of chromosomal DNA replication. Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3' to 5' DNA helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt). In this report, we show that the presence of both 5' and 3' single-stranded tails in DNA helicase substrates is essential for the processive helicase activity of the Mcm complex. The presence of both 5' and 3' tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive helicase activity. The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length. These results support the hypothesis that the Mcm4/6/7 complex can function as a replication helicase.

Cell Cycle Proteins↗
PubMed2001

The role of bacteriologic infection in the etiology of nasolacrimal duct obstruction.

BACKGROUND: In patients with nasolacrimal duct obstruction, the inflammation and fibrosis may be secondary to coexisting infectious colonization within the lumen of the lacrimal sac. We examined the bacterial flora within the lacrimal sac at the sac-duct junction to explore the possibility of a primary bacteriologic etiology of the inflammatory response. METHODS: The study included 114 consecutive patients (132 sides) with epiphora undergoing routine external dacryocystorhinostomy (DCR) at a tertiary care hospital in Toronto between November 1999 and April 2000. A lacrimal sac tissue biopsy specimen was acquired intraoperatively from the inferior posterior lacrimal sac near the sac-duct junction. Microbiologic analysis was carried out to determine the presence and identification of infectious organisms. RESULTS: Culture gave positive results in 51 patients (44.7%) (41.7% of specimens). A total of 65 isolates were cultured, of which 78.5% were gram-positive bacteria and 21.5% gram-negative bacteria. Of the gram-positive organisms 76.5% were Staphylococcus sp. The presence of a positive culture result was independent of a history of dacryocystitis or the presence of a mucocele. INTERPRETATION: Using direct biopsy methods, we found culture-positive lacrimal sac specimens in a large proportion of patients undergoing DCR surgery. These organisms were found to be present in patients with and without a history of infection.

Bacterial Typing Techniques↗
PubMed2001

Physical and functional interactions of human DNA polymerase eta with PCNA.

Human DNA polymerase eta (hPoleta) functions in the error-free replication of UV-damaged DNA, and mutations in hPoleta cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPoleta is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPoleta with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPoleta inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPoleta, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K(m) for the incoming nucleotide.

Amino Acid Sequence↗
PubMed2000

Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.

Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

Antigens, Polyomavirus Transforming↗
PubMed2000

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Amino Acid Sequence↗
PubMed2000

Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe.

Minichromosome maintenance (Mcm) proteins 2-7 are highly conserved in eukaryotes and play an essential role in DNA replication. Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccharomyces pombe using the baculovirus expression system. The simultaneous expression of all six of the Mcm proteins, as well as different combinations of these proteins, yielded several stable complexes that included the heterohexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer. The purification and characterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities. Consistent with these results, the interaction of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the disassembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase activity. These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulation of the activity of this complex.

Base Sequence↗
PubMed2000

Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation.

Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.

Cell Cycle↗
PubMed2000

Fidelity of eucaryotic DNA polymerase delta holoenzyme from Schizosaccharomyces pombe.

The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.

Base Sequence↗
PubMed2000

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum.

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid MTH: ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K(m) 1.1 microM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD(+) cannot. MTH: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase-adenylate intermediate (step 1) and hence cannot seal a 3'-OH/5'-PO(4) nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase-adenylate formation, but defective in activating the nick via formation of the DNA-adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA-adenylate, could be elicited for the wild-type MTH: ligase by inclusion of calcium as the divalent cation cofactor. MTH: ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that MTH: ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.

Adenosine Triphosphate↗
PubMed2000

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway.

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.

DNA Damage↗
PubMed2000

A unique organization of the protein subunits of the DNA polymerase clamp loader in the archaeon Methanobacterium thermoautotrophicum deltaH.

Replication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases delta and epsilon. Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon, Methanobacterium thermoautotrophicum DeltaH. In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits. The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex. The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E. coli. Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3 protomers. Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation. The importance and roles of RFC and PCNA in M. thermoautotrophicum DeltaH replication are discussed.

Adenosine Triphosphatases↗
PubMed2000

Structure and activity associated with multiple forms of Schizosaccharomyces pombe DNA polymerase delta.

DNA polymerase delta (Pol delta) isolated from Schizosaccharomyces pombe (sp) consists of at least four subunits, Pol3, Cdc1, Cdc27, and Cdm1. We have reconstituted the four-subunit complex by simultaneously expressing these polypeptides in baculovirus-infected insect cells. The properties of the purified cloned spPol delta were identical to the native spPol delta isolated from S. pombe cells. In addition, we also isolated a three-subunit complex containing Pol3, Cdc1, and Cdm1. Both three- and four-subunit complexes required replication factor C and proliferating cell nuclear antigen for DNA replication. However, in the presence of low levels of polymerase complexes, the three-subunit complex was less efficient than the four-subunit complex in supporting DNA replication. The inefficient synthesis of DNA by the three-subunit complex can be remedied by the addition of Cdc27, the subunit missing in the three-subunit complex. Gel filtration analysis demonstrated that the three-subunit complex is a monomer of the heterotrimer (Pol3, Cdc1, and Cdm1) and that the four-subunit complex is a dimer of the heterotetramer (Pol3, Cdc1, Cdc27, and Cdm1), similar to the structure of native spPol delta. We have further shown that Cdc1 and Cdc27 interact to form a heterodimeric complex. Gel filtration studies indicate that the structure of this complex is dimeric. These observations suggest that the Cdc27 subunit may play an important role contributing to the dimerization of Pol delta.

Cell Cycle Proteins↗
PubMed2000

Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure.

Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed.

Base Pairing↗
PubMed1999

The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains DNA helicase activity.

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508-24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum DeltaH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric ( approximately 10(3) kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3'-to-5' ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.

Adenosine Triphosphatases↗