Immunoaffinity-purified DNA polymerase alpha displays novel properties

The purification and characterization of a novel and more intact form of the DNA polymerase alpha-primase complex from calf thymus are described. The polymerase-primase was enriched 10,000-fold to apparent homogeneity by chromatography on phosphocellulose, heparin-Sepharose, and an immobilized anti-human DNA polymerase alpha monoclonal antibody [SJK287-38; Tanaka, S., Hu, S., Wang, T. S.-F., & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390]. A quantitative elution from the antibody column was achieved by shifting the pH from neutrality to between 12.5 and 13. From 1 kg of calf thymus, the procedure yields 1-2 mg of polymerase-primase with a specific activity of 30,000-40,000 units/mg for the polymerase and 15,000-20,000 units/mg for the primase. The complex sediments at 9 S through a sucrose gradient and exhibits a Stokes radius of 6.0 nm, yielding a native molecular mass of 335,000. Denaturing gel electrophoresis of the complex gives bands of Mrs 180,000, 155,000, 148,000, 73,000, 59,000, and 48,000 with a relative abundance of the two smallest subunits. Primase activity was partially resolved from the complex by centrifugation through sucrose gradients. The primer-forming activity was found to be associated with the Mr 59,000 and 48,000 polypeptides. In contrast to conventional preparations, the immunopurified polymerase displays several features which show it is the most intact form of the enzyme known to date. The deoxynucleoside triphosphate Km values are all within the range of 0.6-0.9 microM. The Km for binding to a single RNA primer on M13 DNA is 3.5 nM; the Ki for nonspecific binding to unprimed DNA is 70 microM (nucleotide).(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA polymerase alpha-DNA primase from human lymphoblasts

The DNA polymerase alpha-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458-8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195,000 and 180,000 for the DNA synthesizing alpha-subunit, of 68,000 for the beta-subunit, and of 55,000 and 48,000 for the primase-carrying gamma- and delta-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the alpha-subunit, at 5.5 for the beta-subunit, and at 7.5 and 8.0 for the gamma- and delta-subunit, respectively. The purified polymerase-primase converted single-stranded phi X174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3-10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.     

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.     

Human Cdc45 is a proliferation-associated antigen

Cell division cycle protein 45 (Cdc45) plays a critical role in DNA replication to ensure that chromosomal DNA is replicated only once per cell cycle. We analysed the expression of human Cdc45 in proliferating and nonproliferating cells. Our findings show that Cdc45 protein is absent from long-term quiescent, terminally differentiated and senescent human cells, although it is present throughout the cell cycle of proliferating cells. Moreover, Cdc45 is much less abundant than the minichromosome maintenance (Mcm) proteins in human cells, supporting the concept that origin binding of Cdc45 is rate limiting for replication initiation. We also show that the Cdc45 protein level is consistently higher in human cancer-derived cells compared with primary human cells. Consequently, tumour tissue is preferentially stained using Cdc45-specific antibodies. Thus, Cdc45 expression is tightly associated with proliferating cell populations and Cdc45 seems to be a promising candidate for a novel proliferation marker in cancer cell biology.     

Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro

Although p48 is the most conserved subunit of mammalian DNA polymerase alpha-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.     

DNA variants in the human RAB3A gene are not associated with autism

Mutation screening of the RAB3A gene in 47 individuals with autism provided no evidence that DNA variants in this gene are associated with autism. Since Rab3a constitutive knockout mice react to novel stimuli with hyperactivity, a further search for association of RAB3A DNA variants with other neurobehavioral disorders such as attention deficit/hyperactivity disorder appears justified.     

The impact of genetic background on neurodegeneration and behavior in seizured mice

We used pilocarpine-induced seizures in mice to determine the impact of genetic background on the vulnerability of hippocampal neurons and associated changes of behavioral performance. The susceptibility of hippocampal neurons to seizure-induced cell death paralleled the severity of the seizures and depended on genetic background. Hippocampal neurons in C57BL/6 mice were most resistant to cell death, whereas they were highly vulnerable in FVB/N mice. The degree of neuronal degeneration in F1 hybrid mice obtained by crossing the two strains was at an intermediate level between the parent strains. Two weeks after the severe seizures, performance in a water-maze place navigation task showed a bimodal distribution. Seventeen of 19 (90%) F1 mice were completely unable to learn while the other two learned reasonably well. Of 28 C57BL/6 mice with similarly severe seizures, six were as strongly impaired as their F1 counterparts (22%). The remaining 22 performed normally, indicating a much lower probability of C57BL/6 mice to be affected. Treated mice showed a deficit of open-field exploration which was strongly correlated with the impairment in the place navigation task and was again more severe in F1 mice. Our results show that the vulnerability of hippocampal neurons to pilocarpine-induced seizures, as well as the associated behavioral changes, depended on genetic background. Furthermore, they confirm and extend our earlier finding that a relatively modest reduction of hippocampal cell death can be associated with dramatic changes of behavioral performance and emphasize the importance of tightly-controlled genetic backgrounds in biological studies.     

The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that is able to grow at in situ temperatures below 0 degrees C. As abundant members of the microbial community in permanently cold marine sediments, D. psychrophila-like bacteria contribute to the global cycles of carbon and sulfur. Here, we describe the genome sequence of D. psychrophila strain LSv54, which consists of a 3 523 383 bp circular chromosome with 3118 predicted genes and two plasmids of 121 586 bp and 14 663 bp. Analysis of the genome gave insight into the metabolic properties of the organism, e.g. the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system, the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc. D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, nine putative cold shock proteins and nine potentially cold shock-inducible proteins. A comparison of D. psychrophila's genome features with those of the only other published genome from a sulfate reducer, the hyperthermophilic archaeon Archaeoglobus fulgidus, revealed many striking differences, but only a few shared features.     

Comparison of prostaglandin F2alpha,bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.