Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Initiation of Adenovirus DNA replication in vitro requires the presence of three viral proteins (pTP, pol, DBP) and two cellular transcription factors, NFI and Oct-1, that stimulate replication more than 100-fold. NFI assists in binding and positioning of the DNA polymerase in the origin whereas Oct-1 changes the structure of origin DNA. Optimal templates contain, in addition to origin sequences, the covalently bound viral terminal protein (TP). This terminal protein stimulates the template activity over 20 fold compared to protein-free templates. To study the way in which TP exerts its function in vitro we devised a novel method to isolate and label a short origin containing fragment in which the TP was bound in a functional form. This fragment replicated very efficiently and could be used for studying the binding of other replication proteins. Employing alpha-chymotrypsin digestion we show that for enhancement of replication in vitro only a small part of TP is required.
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PMID:Adenovirus DNA replication: the function of the covalently bound terminal protein. 129 Dec 41

POU domain proteins constitute a family of eukaryotic transcription factors that exert critical functions during development. They contain a conserved 160 amino acids DNA binding domain, the POU domain. Genetic data have demonstrated that some POU domain proteins are essential for the proliferation of specific cell types, suggesting a possible role in DNA replication. In addition, the ubiquitous POU transcription factor Oct-1 or its isolated POU domain enhances adenovirus DNA replication. Here we compared the DNA binding specificities of POU domain proteins from different subclasses. They exhibit overlapping, yet distinct binding site preferences. Furthermore, purified Pit-1, Oct-1, Oct-2, Oct-6, Oct-4 and zebrafish POU[C] could all stimulate adenovirus DNA replication in a reconstituted in vitro system. Thus, activation appears to depend on a property common to most POU domain proteins. Adenovirus DNA replication is also stimulated by the transcription factor NFI/CTF. In contrast to NFI, the POU domain did not enhance binding of precursor terminal protein-DNA polymerase to the origin nor did it stabilize the preinitiation complex. These results suggest that the POU domain acts on a rate limiting step after formation of the preinitiation complex.
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PMID:POU domain transcription factors from different subclasses stimulate adenovirus DNA replication. 147 98

A ubiquitous mammalian transcription factor, Oct-1 (also known as OTF-1, NF-A1, OBP100, or NFIII), stimulates the initiation of replication of adenovirus DNA, and may also be involved in the activation of some chromosomal replication origins. If this is true, binding sites for Oct-1 should be present within regions responsible for the initiation of DNA replication. In this study such a binding site has been identified within a 340bp fragment that was originally isolated from a minor fraction of DNA associated with a complexed form of DNA polymerase alpha from nonregenerating rat liver, and which shows autonomous replication sequence activity in a transient transfection assay. Northern blot analysis was used to show that Oct-1 mRNA is induced in regenerating rat liver 6-14 h after hepatectomy.
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PMID:Rat DNA sequence associated with a complex form of DNA polymerase alpha in nonregenerating liver interacts with a ubiquitous transcription/replication factor Oct-1. 184 28

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.
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PMID:Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication. 766 96

The bipartite POU domain of transcription factor Oct-1 stimulates adenovirus DNA replication through an interaction with the octamer sequence present in the auxiliary origin. Employing an immobilized in vitro DNA replication system, we show that the POU domain enhances the formation of a pre-initiation complex composed of the viral precursor terminal protein-DNA polymerase (pTP-pol) complex and the origin. To investigate the mechanism of stimulation we have explored protein-protein interactions between the POU domain and the pTP-pol complex. Such an interaction could be detected using a GST-POU fusion protein bound to glutathione-agarose beads. Binding was also observed with the POU homeodomain (POUHD), albeit weaker than with the intact POU domain, but not with the POU specific subdomain. Four point mutations localized in the POUHD were analyzed for pTP-pol binding. Two of these, E22A and E30A, bound pTP-pol equally as well as the wild-type, while the other two, Q24A and E29A, were able to bind 2- to 4-fold better. These mutations are localized in the same region where the HSV transactivator VP16 binds, but did not coincide with the VP16 contacts. A direct correlation between pTP-pol binding and stimulation of DNA replication in vitro was observed for all mutants, suggesting that stimulation by the POU domain is caused by an interaction with the viral pTP-pol complex.
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PMID:The Oct-1 POU domain stimulates adenovirus DNA replication by a direct interaction between the viral precursor terminal protein-DNA polymerase complex and the POU homeodomain. 795 6

Earlier, a number of DNA fragments were identified in the complex form of DNA polymerase alpha. One of them, DARC146, can support autonomous replication in mammalian cells. We have subcloned 146 bp from DARC146 (here called DARC146). This fragment has an ability to replicate autonomously in mammalian cells. This ability permits one to speak about DARC146 as a putative replication origin. From this conclusion, we suggest that all signals for initiation of DNA synthesis are located on the nucleotide sequence under study. Here, we have shown that the nuclear extract contains four polypeptides binding specifically to synthetic oligonucleotides covering the AT-rich region of the DARC146 sequence. The first protein is Oct-1, a nuclear transcription-replication factor. The second protein (named p65) binds to the TCTCTTA site of the DARC146 nucleotide sequence. There are two sites for Oct-1 protein and two sites for p65 in the DARC146 fragment. Octamer motifs and sites for p65 are located tandemly side by side. Moreover, we identified 28kDa polypeptide from nuclear matrix which bound to DARC146. Based upon the data presented, we suggest a hypothetical model of the pre-initiation state of the DARC146 sequence.
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PMID:[DNA fragment DARC146 from a complex form of DNA polymerase alpha contains several nuclear protein binding segments]. 799 Aug 10

Initiation of adenovirus DNA replication in vitro minimally requires the viral TP-DNA template and the precursor terminal protein-DNA polymerase heterodimer (pTP-pol). Optimal initiation occurs in the presence of the cellular transcription factors NFI and Oct-1 and the viral DNA binding protein (DBP). We have studied the influence of these three stimulatory proteins on the kinetics of formation of the pTP-dCMP initiation complex. NFI increases the Vmax of the reaction but does not affect the apparent Km for dC-TP. This indicates that NFI acts by enlarging the amount of active initiation complex in agreement with its stabilizing effect on binding of pTP-pol to the template. Similar kinetic effects were observed for Oct-1. Since Oct-1 does not stabilize binding of pTP-pol to the origin this suggests that Oct-1 increases the rate of pTP-dCMP formation. DBP stimulates the initiation reaction in two ways. First, it moderately increases the Vmax at suboptimal NFI concentrations, which is related to its enhancing effect on binding of NFI to the origin. Second, a much larger stimulation was caused by DBP itself based on a reduction of the Km for dCTP, which was independent of the concentration of pTP-pol or NFI. The Km for dCTP during initiation is lower than during elongation.
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PMID:The adenovirus DNA binding protein effects the kinetics of DNA replication by a mechanism distinct from NFI or Oct-1. 844 75

The adenovirus terminal protein (TP) is covalently linked to the 5' ends of the adenovirus genome and enhances DNA replication in vitro by increasing template activity. To study the effect of TP in more detail we isolated short origin fragments containing functional TP using anion exchange chromatography. These fragments were highly active as templates for DNA replication in a reconstituted system. Employing band-shift assays we found that the affinity of the precursor terminal protein-DNA polymerase complex for the TP-containing origin was increased 2 to 3-fold. Binding affinities of two other replication stimulating proteins, NFI and Oct-1, were not influenced by the terminal protein. Upon DNaseI footprinting we observed, unexpectedly, that the breakdown pattern had changed at various positions in the origin, notably in the area 3-6 and 41-51 by the presence of TP. Some differences in the footprint pattern of NFI and Oct-1 were also found. Our results indicate that TP induces subtle changes in the origin structure that influence the interaction of other replication proteins.
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PMID:The adenovirus terminal protein influences binding of replication proteins and changes the origin structure. 850 26

Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.
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PMID:The Oct-1 POU homeodomain stabilizes the adenovirus preinitiation complex via a direct interaction with the priming protein and is displaced when the replication fork passes. 901 82

Motifs for sequence specific-protein-DNA interactions, such as helix-turn-helix, zinc finger and leucine zipper, are now better understood as a result of extensive studies of three-dimensional (3D) structures of transcription factors. On the other hand, little attention has been paid to motifs for sequence nonspecific binding, namely DNA-phosphate binding. To address the question whether different transcription factors and DNA manipulation enzymes, that is enzymes that work on DNA, share a similar mode of phosphate binding, we surveyed interactions between DNA and protein module, a structural unit of a globular protein. We analyzed the modular organization of DNA polymerase beta and found that residues making contact with DNA phosphates were localized to five modules. Structural comparison of these phosphate-binding modules against others in transcription factors and DNA manipulation enzymes revealed that DNA polymerase beta, the Oct-1 POU domain, 434 Cro and the Arc repressor have a phosphate-binding module with 3D structures similar to one another. This newly detected module, the phosphate-binding helix-turn-helix (pbHTH) module, named for its function and 3D structure, interacts with DNA by (i) making hydrogen bonds between a DNA phosphodiester oxygen and an amino hydrogen of the main chain located at the N-terminus of a C-terminal alpha-helix, and (ii) making electrostatic interactions between DNA phosphates and side chains of lysine or arginine. Finding structurally and functionally similar phosphate-binding units in different transcription factors and DNA manipulation enzymes suggests that shuffling of modules is not limited to the DNA base-recognition motif. Phosphate-binding modules are apparently also shuffled in DNA-binding proteins.
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PMID:Repetitive use of a phosphate-binding module in DNA polymerase beta, Oct-1 POU domain and phage repressors. 1022 61


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