Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in Vitro

A rapid and quantitative nitroceUulose filter-binding assay is described for the detection of nuclear factor I, aI HpLa ceUl sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication.In this apsay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolyrneric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment contiinig a functionl nuclear factor I binding te compared with a control DNA fragment to which nuclear factor I-dd not bind specificaly. This specifi.c DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay alowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells an4 reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA bipding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That tihis fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its functiop in the initiation of adenovirus DNA replication.Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol Qf nuclear factor I-binding site.The efficient replication of adenovirus DNA can be accomplished in vitro by five purified proteins in the presence of the appropriate cofactors (for ,a review, see reference 43). Three of these proteins, adenovirus DNA polymerase (26, 45), DNA-binding protein (DBP) (13,22,38,46), and terminal protein precursor (pTP) (6,8,11,27,44,48), are viral encoded. The two remaining proteins required to fully reconstitute adenovirus replic4tion in vitro are the HeLa cell proteins nuclear factors I (NFI) and II.Nuclear factor II appears to be required for the completion but not the initiation of DNA replication in vitro because, in the absence of nuclear factor II, DNA replication initiates normally but terminates randonily, approximately one-third the way into the genome. Purified preparations of nuclear factor II contain topoisomerase I activity. Furthermore, topoisomerase I purified from either HeLa cells or calf thymus can completely substitute for factor II. Thus, it seems likely that factor II is a type I topoisomerase (33...

Section: Discussionmentioning

confidence: 99%

“…Three of these proteins, adenovirus DNA polymerase (26, 45), DNA-binding protein (DBP) (13,22,38,46), and terminal protein precursor (pTP) (6,8,11,27,44,48), are viral encoded. The two remaining proteins required to fully reconstitute adenovirus replic4tion in vitro are the HeLa cell proteins nuclear factors I (NFI) and II.…”

mentioning

confidence: 99%

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

Diffley¹,

Stillman²

1986

“…In addition, chymotryptic cleavage of wt DBP produces a 44-or 34-kilodalton carboxyl-terminal fragment, or both, which retains full activity in the in vitro elongation of adenovirus DNA (5,15). Thus, like the SV40 T antigen, the DBP exhibits separate functional domains.…”

mentioning

confidence: 99%

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Klessig¹,

Brough²,

Cleghon³

1984

The DNA-bindihg protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central iole in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by IBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DB3P is totic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50-to 200-fold, and within 8 h its synthesis fromb the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.A number of multifunctional proteins in euearyotes have been defined by,a combination of genetic and biochemical studies. One of the best characterized of these is the simian virus 40 (SV40) large T antigen, which exhibits separate functional domains as discerned by mapping of sets of mutations which alter one but not another of the various protein functions.The human adenovirus 72-kilodalton DNA-binding protein (DBP) also plays several independent roles during the infectious cycle of this virus. Several mutants with temperature sensitive (tsj alterations in the DBP have been indispensable in defining several of the functions of this protein. The ts alterations at the nonpermissive temperature diminish the single-strand, DNA-binding activity of the protein in vitro (52) and inhibit DNA replication in vivo (13, 47, 53) and in vitro (20). Normal turn-off of viral early genes during the late phase of the lytic cycle is also disrupted at the nonpermissive temperature in cells infected with the ts mutant (6,9,10,37,38). The DBP directly affects early gene turn-off as well as DNA replication, since inhibition of viral DNA synthesis with either drugs or ts lesions in other viral early genes does not affect early gene expression (9, 10). In addition, at the nonpermissive temperatui*, the ts mutants transform rat cells in culture with an enhanced frequency compared with wild-type (wt) virus (16,54).A second class of mutants which greatly enhance the ability of this human virus to grow in monkey cells defines yet a second set of functions of DBP (1,26,29). In monkey cells infected with wt human adenovirus, the viral early genes are properly expressed (3), and viral DNA replication occurs no...

“…The results of our initial investigations have demonstrated a strong sequence relationship between the promoter region of the human B isozyme and that of the adenovirus E2E gene (4). The E2E region of the adenovirus encodes a 72-kilodalton (kDa), single-stranded, DNA-binding protein which is involved in virus replication (9). The expression of this gene is highly dependent on the adenovirus oncogenic products of the Ela region (1).…”

mentioning

confidence: 99%

Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a.

Kaddurah‐Daouk¹,

Lillie²,

Daouk³

et al. 1990

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator Ela. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the Ela region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by Ela domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of Ela with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.The creatine kinases (CKs) are a family of enzymes which are involved in the maintenance of ATP at sites of cellular work (2). Three cytoplasmic isoenzymes of CK have been identified in human tissue. These isoenzymes are dimeric molecules with two dissociable subunits designated as M (muscle) or B (brain) type that can reassociate to form the electrophoretically distinct MM, BB, or MB isotype. The amino acid sequences of these two subunits are encoded by distinct genes (28)(29)(30). The basis for the tissue-specific expression of these isozymes is not known. The MM dimer is the major form in skeletal muscle and myocardium; the MB exists in myocardium, while the BB isozyme exists in embryonic and many other tissues at low levels, but most predominantly in the brain and neuronal tissues. The brain isozyme has specific features which form the background for our investigations. For example, elevated