Biomedical subjects

N Applegren

Publications and source records attributed to N Applegren.

4 recordsLinked to original sources

PubMed1996

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

3T3 Cells↗

PubMed1995

Further characterization of the human cell multiprotein DNA replication complex.

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.

Blotting, Western↗

PubMed1993

Antihelicase action of DNA-binding anticancer agents: relationship to guanosine-cytidine intercalator binding.

DNA-binding antibiotics such as intercalators, narrow groove binders, and other substances modify duplex DNA, making it an altered substrate for DNA helicases. The intercalators daunorubicin, actinomycin D, echinomycin, and elsamicin, the narrow groove binders distamycin and mithramycin, and the plant toxin teniposide, each representing a different chemical class, block SV40 large T antigen DNA helicase action with IC50 values ranging from 4 x 10(-8) to 2 x 10(-6) M. A partially purified human HeLa cell DNA helicase is also potently blocked by daunorubicin, distamycin, and teniposide. Because eukaryotic cells contain helicases of varying abundance, specificity, and type, this site of action for DNA-binding antibiotics may help explain antibiotic potency and specificity for DNA or RNA inhibition. The antihelicase effect of the antibiotic-double-stranded DNA complex may be central to the anticancer activities of these substances. An additional interesting correlation is the antihelicase action of DNA-intercalating antibiotics and their DNA-binding preference for G-C base pair sites. The G-C base pair binding preference of the intercalating antibiotics may result from evolutionary selection because of the higher G-C binding stability, compared with A-T binding stability. The combination of the higher base pair stability at G-C regions and increased duplex DNA stability induced by intercalating antibiotic yields a total additive stability of the intercalator-G-C base pair complex that resists helicase action.

Animals↗

PubMed1992

The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16.

Rta, encoded by Epstein-Barr virus (EBV), is a potent activator of transcription via enhancer sequences located upstream of several viral genes. To identify the domains of Rta that facilitate transcription by interacting with cellular transcription factors, different segments of Rta were linked to the DNA binding domain of yeast transactivator GAL4 (residues 1 to 147). These GAL4-Rta fusion proteins were tested in transfected cells for their ability to activate the adeno E1b promoter with an upstream GAL4 DNA binding site. The acidic C-terminal domain of Rta (amino acids 520 to 605) was a potent activator but behaved differently from VP16 in dose-response and competition experiments. A subterminal domain of Rta (amino acids 416 to 519) linked to GAL4 had weak activation activity. Deletion of these domains from native Rta showed that the C-terminal domain was required for transactivation, but the subterminal domain was required only in B cells. The C-terminal activation domain of Rta contains a pattern of positionally conserved hydrophobic residues shared with VP16 and other transactivators. Substitution of several conserved hydrophobic amino acids in Rta severely impaired transactivation. The improtance of hydrophobic residues was further substantiated by comparing EBV Rta with that of herpesvirus saimiri, which revealed little sequence similarity except for a few acidic residues and the positionally conserved hydrophobic amino acids. The C-terminal domain of EBV Rta contains three partially overlapping copies of this hydrophobic motif. Mutational analysis indicated that all three copies were required for full activity. However, two of the three copies appeared to be sufficient to produce full activity on a target promoter with multiple binding sites, suggesting that these motifs are functional subdomains that can synergize.

Amino Acid Sequence↗