Replicase properties

The a-helical coiled coil-based screening system already provided a wide variety of information about the interactions of fluorinated amino acids within hydrophobic and hydrophilic protein environments. Investigations on the thermal stability as well as the replicase activity have both emphasized the orthogonal properties of fluorinated aliphatic amino acid side chains. The term orthogonal in this context has been chosen by us to demonstrate that they are in fact hydrophobic... 

The Chemistry of Nucleic Acid Biosynthesis Describe three properties common to the reactions catalyzed by DNA polymerase, RNA polymerase, reverse transcriptase, and RNA replicase. How is the enzyme polynucleotide phos-phorylase similar to and different from these three enzymes ... 

Table 4 summarizes the properties of the different DNA polymerases which have been purified from archaebacteria. It is clear from studies performed in vivo that halophiles and some methanogens have at least one aphidicolin-sensitive DNA polymerase involved in DNA replication (most probably a replicase) on the other hand, only one DNA polymerase, either sensitive or resistant to aphidicolin, has been detected in various archaebacteria (with the exception of H. halobium). What are the phylogenetic relationships between these aphidicolin-resistant and -sensitive enzymes In collaboration with F. Lottspeich, we obtained the amino-acid sequences of several peptides from S. acidocaldarius DNA polymerase (resistant) and we observed that these sequences are present in the primary structure of S. solfataricus DNA polymerase (sensitive). This suggests that aphidicolin-resistant and aphidicolin-sensitive DNA polymerases detected in archaebacteria are homologous. It remains to determine whether aphidicolin-resistant enzymes contain the amino-acid motifs typical for DNA polymerases of the B family. 

Infection of animal cells with a picomavirus results in the formation of an RNA dependent RNA polymerase (replicase) which is apparently responsible for the biosynthesis of the viral RNA genome. The discovery of the picomavirus replicase (l-3) and similar enzymatic activities in E, coli infected with RNA bacteriophages (4-6) were made about fifteen years ago. Today, there is detailed knowledge of the structure and properties of the replicases of bacteriophage Qg (7i 8) and f2 (9)j but only partial infoimation on the picomavirus replicase (10-14) This rather slow progress is due mainly to the difficulties encountered in the isolation of a stable ENA dependent replicase from a eukaryotic cell-vims system. 

Although a stable preparation of a picornavirus replicase has not yet been obtained, it may be instructive for fut ire work to summarize some of the experience gained so far in attempts to purify and study the properties of the replicase of encephalomyocarditis (EMC) virus. We will therefore describe in some detail our experimental approach to the isolation of an RNA-dependent polymerase from baby hamster kidney cells (BHK 21) infected with EMC virus, and also results of experiments aimed to identify EMC virus-specific proteins in the piirified enzyme (15) ... 

The work carried out so far on the EMC virus replicase provides a method for the isolation of minute quantities of an unstable RNA dependent replicase which allowed one to conduct a preliminary study of some of the properties of the enzyme. However, information on the ezyme s subunit composition, its mode of action, and on possible additional factors that may determine its specificity toward an EMC vims RNA template will require a stable enzyme preparation at a higher level of purity. There is also a growing feeling that the elucidation of the mechanism of biosynthesis of the picomaviral RNA will probably depend on an understanding of the possible functional relationships between the viral RNA replication complex and the smooth membranes in which it is enclosed. 

PSF specifically stimulated the primase activity and, thus, the DNA replicase activity of purified DNA polymerase a-primase. As shown in Fig. 1, the primer synthesis by this enzyme was completely dependent on template, required appropriate concentration of deoxyribonucleoside triphosphate and was markedly stimulated by a very low concentration of PSF (more than 20-fold stimulation was observed at 10 ng/50 pi of PSF). The factor was also effective in stimulating the replicase activity supported by single stranded calf thymus or 0X 174 DNA as template, indicating that primer synthesis in these reactions was also stimulated. However, PSF is not primase itself since the factor neither shows any primer synthesis by itself nor does it stimulate the activity of E. coli DNA polymerase I with poly (dT) as template. A factor with properties similar to PSF has been found in mouse tissue by Yagura, Kozu, and Seno (8) but was not detected in other vertebrates tested by them (9). However, the molecular component of their factor was significantly smaller (63 kDa) than the one described here (8). 

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