Expression, purification, and characterization of DNA polymerases involved in papovavirus replication

• Published in: Methods in enzymology Vol. 275; p. 133
• Main Authors: Malkas, L H, Hickey, R J
• Format: Journal Article
• Language: English
• Published: United States 1996
• Subjects: Animals; Cell Line; Chromatography, Affinity - methods; Chromatography, Gel - methods; Chromatography, Ion Exchange - methods; Cloning, Molecular - methods; DNA Polymerase II - isolation & purification; DNA Polymerase II - metabolism; DNA Polymerase III; DNA Primase; DNA Replication; DNA-Directed DNA Polymerase - biosynthesis; DNA-Directed DNA Polymerase - isolation & purification; DNA-Directed DNA Polymerase - metabolism; HeLa Cells; Humans; Indicators and Reagents; Mice; Papillomaviridae - physiology; Polyomaviridae; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA Nucleotidyltransferases - isolation & purification; RNA Nucleotidyltransferases - metabolism; Simian virus 40 - enzymology; Simian virus 40 - physiology; Templates, Genetic; Virus Replication
• ISSN: 0076-6879
• DOI: 10.1016/S0076-6879(96)75011-5

In recent years, work from a large number of laboratories has greatly expanded our knowledge of the biochemical characteristics and the genetic structure of the DNA polymerases used during papovavirus DNA replication. The development of in vitro DNA replication systems for both SV40 and polyoma virus has been paramount in facilitating the development of the current models describing how DNA polymerase alpha and delta function to replicate the genomes of these two viruses. Our studies have demonstrated that the proteins recognized to be essential for both in vitro SV40 and polyoma viral origin-dependent DNA synthesis can be isolated from cells as an intact complex. We have shown that the human cell MRC closely resembles the murine cell MRC, in both its protein composition and its fractionation and chromatographic profile. In addition, our data regarding both the human and the murine MRC support the dipolymerase model proposed from in vitro DNA replication studies using reconstituted assay systems. In addition, analysis of the nucleotide sequence of the genes encoding DNA polymerase alpha and delta has revealed that the amino acids encoded by several regions of these two genes have been rigorously maintained across evolutionary lines. This information has permitted the identification of protein domains which mediate the complex series of protein-protein interactions that direct the DNA polymerases to the cell nucleus, specify complete or partial exonuclease active sites, and participate in the interaction of each DNA polymerase with the DNA template. Expression studies examining each of the genes encoding DNA polymerase alpha and delta clearly indicate that both DNA polymerases are cell cycle regulated and undergo a dramatic induction in their expression when quiescent cells are stimulated to enter the cell cycle. This is in contrast to the two- to three-fold upregulation in the level of expression of these two genes when cycling cells cross the G1/S boundary. In addition, both proteins are phosphorylated in a cell cycle-dependent manner, and phosphorylation appears to be mediated through the action of a cdc2-dependent protein kinase. Despite all of this new information, much remains to be learned about how papovavirus DNA replication is regulated and how these two DNA polymerases act in vivo to faithfully copy the viral genomes. Studies have yet to be performed which identify all of the cellular factors which potentially mediate papovavirus DNA replication. The reconstituted replication systems have yielded a minimum number of proteins which are required to replicate SV40 and polyoma viral genomes in vitro. However, further studies are needed to identify additional factors which may participate in each step of the initiation, elongation, and termination phases of viral genome replication. As an example, models describing the potential role of cellular helicases, which are components of the MRC isolated from murine and human cells, have yet to be described. It is also conceivable that there are a number of other proteins which serve to attach the MRC to the nuclear matrix, stimulate viral DNA replication, and potentially regulate various aspects of the activity of the MRC throughout viral DNA replication. We are currently working toward characterizing the biochemical composition of the MRC from both murine and human cells. Our goals are to identify all of the structural components of the MRC and to define the role of these components in regulating papovavirus and cellular DNA replication. We have also begun studies to visualize the spatial organization of these protein components within the MRC, examine the regulatory processes controlling the activity of the various components of the MRC, and then develop this information into a coherent picture of the higher order structure of the MRC within the cell nucleus. We believe that this information will enable us to develop an accurate view of the detailed processes mediating both pa