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M D Challberg

Publications and source records attributed to M D Challberg.

At least 19 recordsLinked to original sources
PubMed2001

Assembly of the human origin recognition complex.

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.

DNA-Binding Proteins↗
PubMed1997

Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding.

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase composed of the products of the UL5, UL52, and UL8 genes. UL5 possesses six motifs conserved among superfamily 1 of helicase proteins. Substitutions of conserved residues in each motif abolishes DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). Purified UL5.52 harboring a Gly to Ala change in motif V retains primase and helicase activities in vitro but exhibits a higher KM for single-stranded DNA and lower DNA-dependent ATPase activity (Graves-Woodward, K. L., and Weller, S. K. (1996) J. Biol. Chem. 272, 13629-13635). We have purified and characterized six other subcomplexes with residue changes in the UL5 helicase motifs. Each variant subcomplex displays at least wild type or greater levels of primase and DNA binding activities, but all are defective in helicase activity. Mutations in motifs I and II exhibit profound decreases in DNA-dependent ATPase activity. Mutations in motifs III-VI decrease DNA-dependent ATPase activity 3-6-fold. Since mutations in motifs III, IV, V, and VI do not eliminate ATP hydrolysis or DNA binding, we propose that they may be involved in the coupling of these two activities to the process of DNA unwinding. This analysis represents the first comprehensive structure-function analysis of the conserved motifs in helicase superfamily 1.

Adenosine Triphosphatases↗
PubMed1995

The stoichiometry of binding of the herpes simplex virus type 1 origin binding protein, UL9, to OriS.

A number of studies have demonstrated that the herpes simplex virus type 1 (HSV-1) UL9 protein, which is a homodimer in solution, binds to two high affinity binding sites in each origin of replication. Interaction between the proteins bound at the two sites leads to the formation of a complex nucleoprotein structure. The simplest models for this binding interaction predict two possible binding stoichiometries: 1) one UL9 dimer is bound at each site; or 2) one UL9 monomer is bound at each site so that one UL9 dimer occupies both sites. Two recent papers have addressed this issue by using indirect methods to measure the binding stoichiometry. Martin et al. (Martin, D. W., Muñoz, R. M., Oliver, D., Subler, M. A., and Deb, S. (1994) Virology 198, 71-80) reported that a monomer of UL9 binds to a single high affinity site, and Stabell and Olivo (Stabell, E. C., and Olivo, P.D. (1993) Nucleic Acids Res. 21, 5203-5211) concluded that a dimer of UL9 binds to a single high affinity site. We have directly measured the stoichiometry of binding of the carboxyl-terminal DNA binding domain of UL9 (t-UL9) to the origin of replication using a double-label gel shift assay. Using a short synthetic double-stranded oligonucleotide containing a single UL9 binding site, one protein-DNA complex was detected in the gel shift assay, and the molar ratio of UL9 DNA binding domains to DNA binding sites in this complex was determined to be 2.0 +/- 0.1 (n = 13). Using the minimal origin sequence excised from plasmid DNA, two protein-DNA complexes were detected. The binding stoichiometry of the faster migrating complex was 1.8 +/- 0.1 (n = 15), and the stoichiometry of the more slowly migrating band was 3.7 +/- 0.4 (n = 15). The simplest explanation for these data is that UL9 binds to the origin of replication as a homodimer with one dimer bound at both high affinity sites.

Animals↗
PubMed1994

Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity.

The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.

Amino Acid Sequence↗
PubMed1994

Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template.

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.

Animals↗
PubMed1992

Purification and characterization of UL9, the herpes simplex virus type 1 origin-binding protein.

UL9, the origin-binding protein of herpes simplex virus type 1 (HSV-1), has been overexpressed in an insect cell overexpression system and purified to homogeneity. In this report, we confirm and extend recent findings on the physical properties, enzymatic activities, and binding properties of UL9. We demonstrate that UL9 exists primarily as a homodimer in solution and that these dimers associate to form a complex nucleoprotein structure when bound to the HSV origin of replication. We also show that UL9 is an ATP-dependent helicase, capable of unwinding partially duplex DNA in a sequence-independent manner. Although the helicase activity of UL9 is demonstrable on short duplex substrates in the absence of single-stranded DNA-binding proteins, the HSV single-stranded DNA-binding protein ICP8 (but not heterologous binding proteins) stimulates UL9 to unwind long DNA sequences of over 500 bases. We were not able to demonstrate unwinding of fully duplex DNA sequences containing the HSV origin of replication. However, in experiments designed to detect origin-dependent unwinding, we did find that UL9 wraps supercoiled DNA independent of sequence or ATP hydrolysis.

Animals↗
PubMed1992

The UL8 subunit of the herpes simplex virus helicase-primase complex is required for efficient primer utilization.

The herpes simplex virus (HSV) type 1 helicase-primase is a three-protein complex, consisting of a 1:1:1 association of UL5, UL8, and UL52 gene products (J.J. Crute, T. Tsurumi, L. Zhu, S. K. Weller, P. D. Olivo, M. D. Challberg, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 86:2186-2189, 1989). We have purified this complex, as well as a subcomplex consisting of UL5 and UL52 proteins, from insect cells infected with baculovirus recombinants expressing the appropriate gene products. In confirmation of previous reports, we find that whereas UL5 alone has greatly reduced DNA-dependent ATPase activity, the UL5/UL52 subcomplex retains the activities characteristic of the heterotrimer: DNA-dependent ATPase activity, DNA helicase activity, and the ability to prime DNA synthesis on a poly(dT) template. We also found that the primers made by the subcomplex are equal in length to those synthesized by the UL5/UL8/UL52 complex. In an effort to uncover a role for UL8 in HSV DNA replication, we have developed a model system for lagging-strand synthesis in which the primase activity of the helicase-primase complex is coupled to the activity of the HSV DNA polymerase on ICP8-coated single-stranded M13 DNA. Using this assay, we found that the UL8 subunit of the helicase-primase is critical for the efficient utilization of primers; in the absence of UL8, we detected essentially no elongation of primers despite the fact that the rate of primer synthesis on the same template is undiminished. Reconstitution of lagging-strand synthesis in the presence of UL5/UL52 was achieved by the addition of partially purified UL8. Essentially identical results were obtained when Escherichia coli DNA polymerase I was substituted for the HSV polymerase/UL42 complex. On the basis of these findings, we propose that UL8 acts to increase the efficiency of primer utilization by stabilizing the association between nascent oligoribonucleotide primers and template DNA.

Adenosine Triphosphatases↗
PubMed1990

Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells.

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.

Animals↗
PubMed1990

The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity.

Genetic experiments have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication, and a number of studies have suggested that these two proteins specifically interact. We have confirmed and extended these findings. The viral DNA polymerase from HSV-1-infected cells has been purified as a complex containing equimolar quantities of the UL30 (Pol, the catalytic subunit) and UL42 polypeptides. Sedimentation and gel filtration analyses of this complex are consistent with the idea that the complex consists of a heterodimer of Pol and UL42. A complex with identical physical and functional properties was also purified from insect cells coinfected with recombinant baculoviruses expressing the two polypeptides. Therefore, the formation of the Pol-UL42 complex does not require the participation of any other HSV-encoded protein. We have compared the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells. The specific activity of the catalytic subunit alone was nearly identical to that of the complex when assayed on activated DNA. When assayed on a defined template such as singly primed M13 DNA, however, the combination of Pol and UL42 utilized fewer primers and formed larger products than Pol alone. Template challenge experiments demonstrated that the Pol-UL42 complex was more highly processive than Pol alone. Our data are consistent with the idea that the UL42 polypeptide is an accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization.

Animals↗
PubMed1989

Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products.

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.

Adenosine Triphosphatases↗
PubMed1989

Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression.

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.

Animals↗
PubMed1988

Inversion events in the HSV-1 genome are directly mediated by the viral DNA replication machinery and lack sequence specificity.

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.

Animals↗
PubMed1988

Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, oriS and oriL. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on oriS, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described previously by Elias et al. [Elias, P., O'Donnell, M. E., Mocarski, E. S. & Lehman, I. R. (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6326] is the product of the UL9 gene.

Animals↗
PubMed1988

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.

Animals↗
PubMed1988

Structures of herpes simplex virus type 1 genes required for replication of virus DNA.

Recently, a method has been developed to identify regions in the genome of herpes simplex virus type 1 (HSV-1) which contain genes required for DNA synthesis from an HSV-1 origin of DNA replication, and seven genomic loci have been identified as representing the necessary and sufficient gene set for such replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). Two of the loci represent the well-known genes for DNA polymerase and major DNA-binding protein, but the remainder had little or no previous characterization. In this report we present the DNA sequences of the five newly identified genes and their deduced transcript organizations and encoded amino acid sequences. These genes were designated UL5, UL8, UL9, UL42, and UL52 and were predicted to encode proteins with molecular weights of, respectively, 99,000, 80,000, 94,000, 51,000, and 114,000. All of these genes had clear counterparts in the genome of the related alphaherpesvirus varicella-zoster virus, but only UL5 and UL52 were detectably conserved in the distantly related gammaherpesvirus Epstein-Barr virus, as judged by amino acid sequence similarity. The sequence of the UL5 protein, and of its counterparts in the other viruses, contained a region closely resembling known ATP-binding sites; this could be indicative, for instance, of a helicase or primase activity.

Amino Acid Sequence↗
PubMed1987

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

Adenoviruses, Human↗
PubMed1986

A method for identifying the viral genes required for herpesvirus DNA replication.

Several laboratories have shown that transfected plasmid DNAs containing either of the two known origins of herpes simplex virus (HSV) DNA replication, oriS or oriL, are replicated in HSV-1-infected cells or in cells cotransfected with virion DNA. I have found that HSV-1 (KOS) DNA digested to completion with the restriction enzyme Xba I is as efficient as intact viral DNA in supporting the in vivo replication of cotransfected plasmids containing oriS. On the basis of this result, several of the Xba I restriction fragments of HSV-1 DNA were cloned into the plasmid vector pUC19, and combinations of cloned DNAs were tested for their ability to supply the trans-acting functions required for HSV origin-dependent replication. A combination of five cloned fragments of HSV-1 can supply all of the necessary functions: Xba I C (coordinates 0.074-0.294), Xba I F (coordinates 0.294-0.453), Xba I E (coordinates 0.453-0.641), Xba I D (coordinates 0.641-0.830), and EcoRI JK (coordinates 0.0-0.086; 0.830-0.865). Transient plasmid replication in this system is dependent on the presence of either oriS or oriL in cis. The plasmid containing Xba I F can be replaced by two smaller plasmids, one of which contains only the gene for the HSV-encoded DNA polymerase, and the other of which contains only the gene for the major DNA binding protein (ICP8). Thus, plasmid DNA replication in this system depends on two of the genes known from genetic studies to be essential for viral DNA replication in infected cells. This system defines a simple complementation assay for cloned fragments of HSV DNA that contain other genes involved in viral DNA replication and should lead to the rapid identification of all such genes.

Animals↗