Biomedical subjects

W W Hurlburt

Publications and source records attributed to W W Hurlburt.

8 recordsLinked to original sources

PubMed2001

Biochemical characterization of osteo-testicular protein tyrosine phosphatase and its functional significance in rat primary osteoblasts.

Rat osteo-testicular protein tyrosine phosphatase (OST-PTP), expressed in osteoblasts and testis, is a receptor-like transmembrane protein with two tandemly repeated phosphatase domains in the cytoplasmic region. In this report, we show that the first domain (CD1) is enzymatically active and appears to be influenced by the catalytically inactive second domain (CD2). The activity of CD1 is specific to phosphorylated tyrosine. Full-length OST-PTP protein expressed in COS cells has a molecular mass of approximately 185 kDa, and immunoprecipitates of this protein using OST-PTP-specific antisera show strong tyrosine phosphatase activity. Expression of OST-PTP mRNA in primary rat calvarial osteoblasts is temporally regulated, and peak expression is found at approximately day 15, which correlated well with the appearance of OST-PTP protein and its associated tyrosine phosphatase activity. Treatment of osteoblasts in culture with antisense oligonucleotides directed against the 5' untranslated region of OST-PTP results in abrogation of differentiation, confirming the functional importance of OST-PTP expression in osteoblast development.

Animals↗

PubMed1997

A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit.

The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.

DNA Helicases↗

PubMed1995

Sequence-dependent primer synthesis by the herpes simplex virus helicase-primase complex.

The herpes simplex virus helicase-primase complex, a heterotrimer of the UL5, UL8, and UL52 proteins, displays a single predominant site of primer synthesis on phi X174 virion DNA (Tenney, D. J., Hurlburt, W. W., Micheletti, P. M., Bifano, M., and Hamatake, R. K. (1994) J. Biol. Chem. 269, 5030-5035). This site was mapped and found to be deoxycytosine-rich, directing the synthesis of a primer initiating with several guanine residues. The size and sequence requirements for primer synthesis were determined using oligonucleotides containing variations of the predominant template. Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely dependent on nucleotide sequence. Conversely, the ATPase activity on oligonucleotide templates was dependent on template size rather than nucleotide sequence. Furthermore, only oligonucleotides containing primase templates were inhibitory in a coupled primase-polymerase assay using phi X174 DNA as template, suggesting that primer synthesis or primase turnover is rate-limiting. Additionally, stimulation of helicase-primase by the UL8 component and that by the ICP8 protein were shown to differ mechanistically using different templates: the UL8 component stimulated the rate of primer synthesis on phi X174 virion DNA and oligonucleotide templates, while ICP8 stimulation occurred only on phi X174 virion DNA.

Adenosine Triphosphatases↗

PubMed1995

Characterization of monoclonal antibodies recognizing amino- and carboxy-terminal epitopes of the herpes simplex virus UL42 protein.

A panel of monoclonal antibodies (MAbs) directed against the herpes simplex virus type 1 (HSV-1) DNA polymerase (Pol) accessory protein, UL42, was developed and characterized. Thirteen different MAbs were isolated which exhibited varied affinities for the protein. All MAbs reacted with UL42 in ELISA, Western blot and immunoprecipitation analyses. Competitive ELISA was used to show that 6 different epitopes within UL42 were recognized by the MAbs. Immunoprecipitation of amino- and carboxy-terminal truncations of UL42 mapped the epitopes to regions containing amino acids 1-10, 10-108, 338-402, 402-460, and 460-477. All but one of these epitopes were outside the minimal active portion of the protein previously mapped to amino acids 20-315. None of these MAbs, alone or in combination, specifically neutralized the ability of UL42 to stimulate Pol activity in vitro. These results are consistent with structure-function studies that showed that N- and C-terminal regions of the UL42 protein, those recognized by the MAbs, are not involved in UL42 function in vitro.

Animals↗

PubMed1994

The UL8 component of the herpes simplex virus helicase-primase complex stimulates primer synthesis by a subassembly of the UL5 and UL52 components.

The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.

Animals↗

PubMed1993

The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain.

Herpes simplex virus type 1 encodes its own DNA polymerase (Pol), the product of the UL30 gene, and a polymerase accessory subunit, the product of the UL42 gene, both of which are required for viral DNA replication. Pol and the UL42 protein associate to form a heterodimeric complex (Pol/UL42) which is more active and has a higher processivity than the Pol catalytic subunit alone. The Pol/UL42 complex has been reconstituted by mixing together highly purified Pol and UL42 subunits obtained from recombinant baculovirus-infected cells. We have used polymerase activity on poly(dA):oligo(dT20), a template that the Pol subunit utilizes with low efficiency, to measure the formation of the Pol/UL42 complex. Our data indicate that the association constant for the Pol/UL42 complex is 1 x 10(8) M-1. Proteolytic digestions of UL42 were performed to determine whether structural domains of UL42 could be disclosed by differential amino acid accessibilities. The ability of these protease-resistant domains to form a functional complex with Pol was determined by measuring their ability to stimulate Pol activity on poly(dA):oligo(dT20). We have found that trypsin digestion of UL42 in the presence of DNA generates protease-resistant fragments of 28K and 8K which co-elute from a MonoQ column and are able to stimulate Pol activity on poly(dA):oligo(dT20). Complex formation of the 28K and 8K tryptic fragments with Pol was also shown by their co-immunoprecipitation with antibody to Pol. It was determined that the 28K fragment of UL42 comprised amino acids 1 to 245 or 1 to 254 of UL42, whereas the 8K fragment started at amino acid 255. Thus, controlled proteolysis of UL42 revealed two closely contiguous structural domains that retained the ability to complex with Pol and stimulate Pol activity.

DNA-Directed DNA Polymerase↗

PubMed1993

Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity.

We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro. Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro. C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro. This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42. Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42. None of the truncations or deletions within Pol affected basal polymerase activity. In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay. These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.

Amino Acid Sequence↗

PubMed1993

Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain.

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.

Amino Acid Sequence↗