Bacteriophage phi29 early protein p17 is conditionally required for the first rounds of viral DNA replication

Taurine is a very important organic osmolyte in most adult cells. Because of this property it has been proposed that large changes in the intracellular content of taurine can osmotically stress the cell, causing changes in its size and shape. This hypothesis was examined by measuring cell dimensions of taurine deficient cardiomyocytes using confocal microscopy. Incubation of isolated neonatal rat myocytes with medium containing 5 mM beta-alanine led to a 55% decrease in intracellular taurine content. Associated with the loss of taurine was a reduction in cell size. Two factors contributed to the change in cell size. First, there was a shift in cell shape, favoring the smaller of the two cellular configurations commonly found in the myocyte cell culture. Second, the size of the polyhedral configuration was reduced after beta-alanine treatment. These same two events also contributed to size reduction in cardiomyocytes incubated with medium containing 30 mM mannitol. Nonetheless, some qualitative differences exist between cells osmotically stressed by increasing the osmolality of the incubation medium and decreasing intracellular osmolality. The results support a role for taurine in the regulation of osmotic balance in the neonatal cardiomyocyte.     

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.     

Genetic exchanges caused by ultraviolet photoproducts in phage lambda DNA molecules: the role of DNA replication

Personality development was examined in two groups of women studied since adolescence who were judged psychologically healthy at age fifty: 1) Independants, whose health improved from forty to fifty, were ambitious and intellectual. 2) Traditionals, healthy at both ages, were gregarious and nurturant. Traditionals showed steady personality growth since adolescence. Independents were constricted at age forty but recovered by fifty. These patterns are compared in terms of the fit between personality and sex role. Traditional personalitites fit conventional feminine roles, accounting for their health throughout the middle years. Independents improved when disengaging from mothering freed them to develop their more assertive skills.     

Increases in the mitochondrial DNA replication and transcription in the remnant liver of rats

A phase I study to determine safety, maximum tolerated dose, and biologic response during multiple once-a-week administration of oral imiquimod, an immune response modifier, was conducted in 12 adults with early human immunodeficiency virus (HIV) infection. All completed the dose-escalation phase of weekly dosing at 100-mg increments and received at least one maintenance dose, 100 mg below the patient's toxic dose, for 12 weeks. Dose-limiting toxicity occurred in 3 patients at 200-mg, 5 at 300-mg, and 3 at 400-mg dose levels. One tolerated the 500-mg dose without dose-limiting toxicity. Dose-limiting toxicities included fatigue, fever, malaise, increased transaminases, hypotension, vomiting, and depression. Seven of 12 completed 12 weeks of maintenance. At > or = 200 mg of imiquimod, all patients had biologic responses, measured by elevations in serum interferon, beta2-microglobulin, and neopterin levels. Imiquimod induced pronounced levels of circulating interferon in asymptomatic HIV-infected persons, with variable effect on virus load.     

phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5' exonucleolysis, is required to interact with the terminal protein

Three amino acid residues highly conserved in most proofreading DNA polymerases, a phenylalanine contained in the Exo II motif and a serine and a leucine belonging to the S/TLx2h motif, were recently shown to be critical for 3'-5' exonucleolysis by acting as single-stranded DNA ligands (de Vega, M., Lázaro, J.M., Salas, M. and Blanco, L. (1998) J. Mol. Biol. 279, 807-822). In this paper, site-directed mutants at these three residues were used to analyze their functional importance for the synthetic activities of phi29 DNA polymerase, an enzyme able to start linear phi29 DNA replication using a terminal protein (TP) as primer. Mutations introduced at Phe65, Ser122, and Leu123 residues of phi29 DNA polymerase severely affected the replication capacity of the enzyme. Three mutants, F65S, S122T, and S122N, were strongly affected in their capacity to interact with a DNA primer/template structure, suggesting a dual role during both polymerization and proofreading. Interestingly, mutant S122N was not able to maintain a stable interaction with the TP primer, thus impeding the firsts steps (initiation and transition) of phi29 DNA replication. The involvement of Ser122 in the consecutive binding of TP and DNA is compatible with the finding that the TP/DNA polymerase heterodimer was not able to use a DNA primer/template structure. Assuming a structural conservation among the eukaryotic-type DNA polymerases, a model for the interactions of phi29 DNA polymerase with both TP and DNA primers is presented.     

Role of the first aspartate residue of the "YxDTDS" motif of phi29 DNA polymerase as a metal ligand during both TP-primed and DNA-primed DNA synthesis

Neurofibromatosis is a systemic disease that often produces striking disfigurement. Orbital manifestations are common and include sphenoid dysplasia with or without infiltration of the periorbital soft tissues. The resultant deficiency of the posterolateral orbital wall may lead to protrusion of the temporal lobe into the orbit, displacement of the globe, and pulsatile exophthalmos. Treatment at our unit has consisted of transcranial orbital reconstruction with bone grafts and periorbital soft-tissue correction. Observation of complete bone graft resorption in one patient prompted an assessment of the Australian Craniofacial Unit's experience with particular attention paid to the stability of operative correction. Of 36 patients with head and neck neurofibromatosis treated during the period from 1981 to 1995, 14 patients underwent transcranial correction of orbital deformities secondary to sphenoid dysplasia. The treatment and outcomes of this transcranial group are reviewed. The most notable finding was that of recurrent globe pulsation in four patients following initial resolution. Computed tomography scans have documented partial to complete bone graft resorption in three of these patients. Titanium mesh is now being utilized to provide a more durable reconstruction.     

Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity

Adequate prenatal care has been linked to improved birth outcomes in general populations but has not been assessed in HIV-infected women. We examined longitudinal claims files and vital statistics records for women in the New York State Medicaid HIV/AIDS data base delivering a singleton from 1985 through 1990. Adequacy of the self-reported number of prenatal visits was assessed by the Kessner index. In logistics models, we estimated the association of prenatal care, illicit drug use, and other maternal characteristics with three outcomes; low birth weight, preterm birth, and small-for-gestational-age. Of 2,254 singletons delivered by this HIV-infected cohort, 28% were low birth weight, 23% were preterm birth, and 20% were small for gestational age. Two-thirds had inadequate prenatal care. Non-drug users had 57 and 26% lower adjusted odds of low birth weight and preterm delivery than drug users. The adjusted odds of low birth weight and preterm birth for women with an adequate number of prenatal visits were, respectively, 48 and 21% lower than for women with inadequate care. Adequate prenatal care was also associated with a 43% reduction in the odds of small-for-gestational-age. An adequate number of prenatal visits by women in this HIV cohort was associated with a significant reduction in all three adverse birth outcomes, but most had inadequate prenatal care. These data support strengthening efforts to bring pregnant, HIV-infected women into care.     

DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.     

Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

Utilization of the same DNA replication origin by human cells of different derivation

In the past, a highly sensitive and efficient method was developed to map DNA replication origins in human cells, based on quantitative PCR performed on nascent DNA samples. This method allowed the identification of a replication origin in the myeloid HL-60 cell line, located on chromosome 19 within an approximately 500 bp segment near the lamin B2 gene [Giacca et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 7119]. The same procedure has now been further simplified and extended to a variety of other exponentially growing human cells of different histological derivation (three neural, one connectival and one epithelial), with a nearly diploid chromosomal content. In all the six cell lines tested, the origin activity within the lamin B2 gene domain was localized to the same region. Furthermore, the lamin B2 origin was also found to be active in stimulated, but not in quiescent, peripheral blood lymphocytes.     

Effect on DNA topology by DnaA protein, the initiation factor of chromosomal DNA replication in Escherichia coli

We examined effects on supercoiled DNA topology of DnaA protein, the initiator protein of chromosomal DNA replication in Escherichia coli. The activity was identified in an analysis of plasmid DNA incubated with DnaA protein and DNA topoisomerase I. In Superose 12 gel filtration chromatography, the activity coeluted with DnaA protein. Incubation of DnaA protein with DNA at temperatures over 24 degrees C was required for this activity, which was observed with either oriC plasmid or the replicative form I of phi X174 with no DnaA box. As binding of ATP or ADP to DnaA protein prevented the activity of DnaA protein on DNA topology, binding of the adenine nucleotide may regulate the activity.