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

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1. Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo. A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J. A. Prendergrast et al., Genetics 124:81-90, 1990; E. A. Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991; A. Rowley et al., Mol. Cell. Biol. 11:5718-5726, 1991), with a spectrum of phenotypes similar to those caused by mutations in the genes encoding histone proteins H2A and H2B (Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991). We show that at the nonpermissive temperature, cdc68-1 mutants arrest as unbudded cells with a 1C DNA content, consistent with a possible role for Cdc68 in the prereplicative stage of the cell cycle. The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism. However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates. Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase alpha may interact with these proteins to gain access to its template or to origins of replication in vivo.
...
PMID:The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein. 919 53

The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.
...
PMID:A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro. 1070 24

HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication and DNA repair, was previously isolated as a complex of a 261-kDa catalytic subunit and a tightly bound 59-kDa accessory protein. Saccharomyces cerevisiae pol epsilon, however, consists of four subunits: a 256-kDa catalytic subunit with 39% identity to HeLa pol epsilon p261, a 80-kDa subunit (DPB2) with 26% identity to HeLa pol epsilon p59, a 23-kDa subunit (DPB3), and a 22-kDa subunit (DPB4). We report here the identification and the cloning of two additional subunits of HeLa pol epsilon, p17, and p12. Both proteins contain histone fold motifs which are present also in S. cerevisiae DPB4 and DPB3. The histone fold motifs of p17 and DPB4 are related to that of subunit A of the CCAAT binding factor, whereas the histone fold motifs found in p12 and DPB3 are homologous to that in subunit C of CCAAT binding factor. p17 together with p12, but not p17 or p12 alone, interact with both p261 and p59 subunits of HeLa pol epsilon. The genes for p17 and p12 can be assigned to chromosome locations 9q33 and 2p12, respectively.
...
PMID:Identification and cloning of two histone fold motif-containing subunits of HeLa DNA polymerase epsilon. 1080 49

The balance between cell differentiation and proliferation is regulated at the transcriptional level. In the cell cycle, the transition from G1 to S phase (G1/S transition) is of paramount importance in this regard. Indeed, it is only before this point that cells can be oriented toward the differentiation pathway: beyond, cells progress into the cycle in an autonomous manner. The G1/S transition is orchestrated by the transcription factor E2F. E2F controls the expression of a group of checkpoint genes whose products are required either for the G1-to-S transition itself or for DNA replication (e.g. DNA polymerase alpha). E2F activity is repressed in growth-arrested cells and in early G1, and is activated at mid-to-late G1. E2F is controlled by the retinoblastoma tumor suppressor protein Rb. Rb represses E2F mainly by recruiting chromatin remodeling factors (histone deacetylases and SWI/SNF complexes), the DNA methyltransferase DNMT1, and a histone methyltransferase. This review will focus on the molecular mechanisms of E2F repression by Rb during the cell cycle and during cell-cycle exit by differentiating cells. A model in which Rb irreversibly represses E2F-regulated genes in differentiated cells by an epigenetic mechanism linked to heterochromatin, and involving histone H3 and promoter DNA methylation, is discussed.
...
PMID:The Rb/chromatin connection and epigenetic control: opinion. 1142 Jul 29

In eukaryotic cells, transcription and replication each occur on DNA templates that are incorporated into nucleosomes. Formation of chromatin generally limits accessibility of specific DNA sequences and inhibits progression of polymerases as they copy information from the DNA. The processes that select sites for initiating either transcription or replication are therefore strongly influenced by factors that modulate the properties of chromatin proteins. Further, in order to elongate their products, both DNA and RNA polymerases must be able to overcome the inhibition presented by chromatin (Lipford and Bell 2001; Workman and Kingston 1998). One way to adjust the properties of chromatin proteins is to covalently modify them by adding or removing chemical moieties. Both histone and non-histone chromatin proteins are altered by acetylation, methylation, and other changes, and the 'nucleosome modifying' complexes that perform these reactions are important components of pathways of transcriptional regulation (Cote 2002; Orphanides and Reinberg 2000; Roth et al. 2001; Strahl and Allis 2000; Workman and Kingston 1998). Another way to alter the effects of nucleosomes is to change the position of the histone octamers relative to specific DNA sequences (Orphanides and Reinberg 2000; Verrijzer 2002; Wang 2002; Workman and Kingston 1998). Since the ability of a sequence to be bound by specific proteins can vary significantly whether the sequence is in the linkers between nucleosomes or at various positions within a nucleosome, 'nucleosome remodeling' complexes that rearrange nucleosome positioning are also important regulators of transcription. Since the DNA replication machinery has to encounter many of the same challenges posed by chromatin, it seems likely that modifying and remodeling complexes also act during duplication of the genome, but most of the current information on these factors relates to regulation of transcription. This chapter describes the factor known variously as FACT in humans, where it promotes elongation of RNA polymerase II on nucleosomal templates in vitro (Orphanides et al. 1998, 1999), DUF in frogs, where it is needed for DNA replication in oocyte extracts (Okuhara et al. 1999), and CP or SPN in yeast, where it is linked in vivo to both transcription and replication (Brewster et al. 2001; Formosa et al. 2001). Like the nucleosome modifying and remodeling complexes, it is broadly conserved among eukaryotes, affects a wide range of processes that utilize chromatin, and directly alters the properties of nucleosomes. However, it does not have nucleosome modifying or standard ATP-dependent remodeling activity, and therefore represents a third class of chromatin modulating factors. It is also presently unique in the extensive connections it displays with both transcription and replication: FACT/DUF/CP/SPN appears to modify nucleosomes in a way that is directly important for the efficient functioning of both RNA polymerases and DNA polymerases. While less is known about the mechanisms it uses to promote its functions than for other factors that affect chromatin, it is clearly an essential part of the complex mixture of activities that modulate access to DNA within chromatin. Physical and genetic interactions suggest that FACT/DUF/CP/SPN affects multiple pathways within replication and transcription as a member of several distinct complexes. Some of the interactions are easy to assimilate into models for replication or transcription, such as direct binding to DNA polymerase alpha (Wittmeyer and Formosa 1997; Wittmeyer et al. 1999), association with nucleosome modifying complexes (John et al. 2000), and interaction with factors that participate in elongation of RNA Polymerase II (Gavin et al. 2002; Squazzo et al. 2002). Others are more surprising such as an association with the 19S complex that regulates the function of the 20S proteasome (Ferdous et al. 2001; Xu et al. 1995), and the indication that FACT/DUF/CP/SPN can act as a specificity factor for casein kinase II (Keller et al. 2001). This chapter reviews the varied approaches that have each revealed different aspects of the function of FACT/DUF/CP/SPN, and presents a picture of a factor that can both alter nucleosomes and orchestrate the assembly or activity of a broad range of complexes that act upon chromatin.
...
PMID:Changing the DNA landscape: putting a SPN on chromatin. 1259 8

The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase beta (pol beta) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates ( approximately 10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing "toward" and "away from" the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol beta is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur "unassisted" in chromatin, whereas downstream factors in this pathway (i.e., pol beta) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.
...
PMID:Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes. 1279 67

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the alpha- and beta-subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the alpha-subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the alpha-subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the gamma-subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.
...
PMID:Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora. 1368 Mar 68

The imprinting control region within the second intron of the mouse Igf2r gene contains a CpG island comprising direct repeats, an imprinting box and the Air antisense promoter which is blocked by the methylation imprint on the active maternal allele. We have investigated the structural features of this DNA, including a mapping of all nucleosome positioning signals within the nucleotide sequence. A discrete series of strong positioning signals distinguished the direct repeat region from the much more diverse positioning capacity of the sequence encompassing the known regulatory elements. At only a few locations did CpG methylation modulate the use of this positioning information. Direct effects upon histone-DNA interactions are therefore unlikely to contribute significantly to the means by which the imprint may establish allele-specific chromatin architecture and determine Air expression. A strand-specific obstruction to DNA polymerase was observed between the repeat and regulatory regions. The same region adopts triple-stranded H-DNA structures in supercoiled DNA, according to pH and divalent cation exposure. Methylation did not modulate the occurrence or form of this structure under the conditions tested. This finding nevertheless adds to the repertoire of potential H-DNA structures found in the vicinity of regulatory sequences-here, in an imprinting context.
...
PMID:Nucleosome positioning signals and potential H-DNA within the DNA sequence of the imprinting control region of the mouse Igf2r gene. 1465 40

Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase epsilon (Pol epsilon) or lacking one of the nonessential and histone fold motif-containing subunits of Pol epsilon, Dpb3 and Dpb4. We also report a novel and putative chromatin-remodeling complex, ISW2/yCHRAC, that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4. Using the single-cell method developed in this study, we demonstrate that without Pol epsilon and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, we reveal that Pol epsilon and ISW2/yCHRAC function independently: Pol epsilon operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators.
...
PMID:Noncompetitive counteractions of DNA polymerase epsilon and ISW2/yCHRAC for epigenetic inheritance of telomere position effect in Saccharomyces cerevisiae. 1467 57

During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.
...
PMID:Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression. 1472 69


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---