Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adenovirus origin of DNA replication is located within the terminal 51 bp of the viral genome and contains three recognizable domains: the minimal origin or "core" and binding sites for the cellular transcription factors NFI (CTF) and NFIII (oct-1, OTF-I). In vivo assays with a series of plasmids containing insertions between the "core" and NFI binding site revealed that a strict spatial arrangement of the NFI binding site relative to the "core" was required for efficient DNA replication. To determine if this strict positional constraint was a result of interactions between genome-bound proteins, we used the DNA-binding domain of NFI immobilized on Sepharose as an affinity matrix to examine binding of the adenovirus DNA polymerase and preterminal protein. Extracts from insect cells infected with baculoviruses expressing the polymerase or preterminal protein were passed over the NFI affinity matrix and bound proteins were eluted. Whereas preterminal protein passed through the column, the DNA polymerase was specifically retained. When extracts containing both preterminal protein and polymerase were passed over the NFI column, both proteins were retained because of the formation of DNA polymerase-preterminal protein heterodimers. Thus, interactions between the DNA binding domain of NFI and the DNA polymerase may serve to direct the DNA polymerase-preterminal protein heterodimer into a preinitiation complex that assembles at the adenovirus origin of DNA replication.
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PMID:Interactions between the adenovirus type 2 DNA polymerase and the DNA binding domain of nuclear factor I. 208

Initiation of adenovirus DNA replication is strongly enhanced by two transcription factors, nuclear factor I (NFI) and nuclear factor III (NFIII/oct-1). These proteins bind to two closely spaced recognition sequences in the origin. We produced NFI and NFIII/oct-1, as well as their biologically active, replication-competent DNA-binding domains (NFI-BD and the POU domain), in a vaccinia virus expression system and purified these polypeptides to apparent homogeneity. By DNase I footprinting and gel retardation, we show that the two proteins, as well as their purified DNA-binding domains, bind independently and without cooperative effects to their recognition sequences. By using a reconstituted system consisting of the purified viral proteins (precursor terminal protein-DNA polymerase complex (pTP-pol) and DNA-binding protein, we show that NFIII/oct-1 or the POU domain stimulates DNA replication in the absence of NFI or NFI-BD and vice versa. When added together, the enhancing effect of the two transcription factors was independent and nonsynergistic. Interestingly, stimulation by NFI or NFI-BD was strongly dependent on the concentration of the pTP-pol complex. At low pTP-pol concentrations, NFI or NFI-BD stimulated up to 50-fold, while at high concentrations, the stimulation was less than twofold, indicating that the need for NFI can be overcome by high pTP-pol concentrations. In contrast, stimulation by NFIII/oct-1 or the POU domain was much less dependent on the pTP-pol concentration. These data support a model in which NFI enhances initiation through an interaction with pTP-pol. Glutaraldehyde cross-linking experiments indicate contacts between pTP-pol and NFI but not NFIII/oct-1. The site of interaction is located in the NFI-BD domain.
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PMID:Transcription factors NFI and NFIII/oct-1 function independently, employing different mechanisms to enhance adenovirus DNA replication. 221 23