David Korn

Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases.

We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady‐state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462‐amino‐acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.

Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases.

The primary sequence of human DNA polymerase α deduced from the full‐length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage ɸ19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl‐terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino‐terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.— Wang, T. S.‐E.; Wong, S. W.; Korn, D. Human DNA polymerase α: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 3: 14‐21; 1989.

Nomenclature of eukaryotic DNA polymerases

vious experiment (Table 1). It could be argued that after a nearly complete depletion of norepinephrine an additional small and undetectable decrease in norepinephrine might surpass some critical threshold and lead to the release of eating. However, if this were true we would also expect this threshold would have been surpassed in at least some of the more than 150 rats in our laboratory which have undergone VNAB destruction via 6-OH-DA injection or electrolytic lesions in previous experiments. In actuality, seven of the eight animals in the combined lesion group ate more food per day than any of the previous VNAB animals studied in this laboratory. Our results would explain why Golds (5) most effective hypothalamic lesions coincided with the distribution of the ventral bundle. Lesions in the medial hypothalamus that destroy portions of the diffuse projections of the VNAB should lead to exceptional hyperphagia such as that observed with dual lesions in the present study. Thus, medial hypothalamic hyperphagia and ventral bundle hyperphagia are separable phenomena; the evidence is: (i) norepinephrine loss caused hyperphagia only at night and less hyperphagia overall, (ii) hypothalamic lesions caused overeating without norepinephrine depletion, and (iii) the two forms of destruction combined produced a level of hyperphagia equal to

Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle.

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

Mechanisms of Catalysis of Human DNA Polymerases α and β

Publisher Summary The chapter discusses a detailed analysis of the mechanisms of catalysis of DNA polymerases α and β was initially generated by the desire to develop a formal enzymological framework within which to evaluate the interactions of the polymerases with candidate replication factors in mechanistically interpretable, model assay systems and was further stimulated by two sets of prior observations: the studies of the patterns of primer-template utilization by these enzymes, and some curious and perplexing results encountered in the examination of the effects of spermidine on the reactivity of DNA polymerase α. The studies summarized in this chapter contribute substantially to the elucidation of some of the mechanisms and specific molecular signals that govern the interaction of DNA polymerases α and β with their nucleic acid substrates. These insights are of interest for at least two reasons. First, in the absence of an exploitable library of mammalian replication/repair mutants, the enzymological properties that have been documented define a mechanistic framework within which to identify and characterize putative accessory replication factors that might act either to modify primer-template structure or to modulate the inherent catalytic properties of the polymerases themselves. The mechanistic elucidation of the stimulation of DNA polymerase α by spermidine provides a prototype for studies of this sort. Second, although it is presumed that the participation of both polymerases α and β in chromosome replication and repair must involve their interaction with a number of additional proteins, the inherent properties of the purified polymerase proteins would appear to be particularly appropriate to the putative roles of these enzymes in these complex and highly regulated processes.

Enzymological Characterization of Human DNA Polymerases-α and β

During the past few years, we have described the results of an extensive series of investigations of the enzymological properties of essentially homogeneous preparations of human DNA polymerases-α and β1-3 that are devoid of contaminating or associated endo- or exodeoxyribonuclease activities4,5. These studies have demonstrated a number of striking differences between the two enzymes with respect to their ability to catalyze deoxynucleotide incorporation on a variety of defined, natural and synthetic DNA primer-templates4-8 and have suggested that there might be equally profound differences, possibly of physiological significance, in the nature of the specific molecular signals that regulate their catalytic interactions with nucleic acids. To pursue these observations in the face of exceedingly limited quantities of purified enzyme protein, we have successfully exploited the power of classical steady-state kinetics methodology to illuminate many of the key features of the polymerase-primer-template interaction and to gain substantial new insights into the fundamental mechanisms of polymerase catalysis. In performing these studies, we have employed direct sedimentation binding assays and novel methods of analysis of polymerase products synthesized on DNA molecules of known sequence to obtain, in every instance, direct physical corroboration of the principal conclusions derived from the kinetics experiments. We have thus been able to provide important reassurance of the validity of the kinetics interpretations and obviate much of the concern that might appropriately arise from the indirect nature of kinetics analyses, particularly in complex systems.

Structure and Catalytic Properties of Human DNA Polymerases α and β

In this paper we shall describe some of our recent studies on the structure and catalytic properties of DNA polymerases a and 8 (1) that we have purified from cultured human KB cells and from adult human livers. The principal topics of discussion will be the definition of the polypeptide composition of near homogeneous KB cell DNA polymerase a, the problem of the intracellular localization of polymerase a, and the results of our initial studies on the specific in Vitro replication of human D-loop mitochondrial DNA (mtDNA) by near homogeneous human DNA polymerase

Monoclonal antibodies against human DNA polymerase-α do not cross-react with glucocorticoid receptors☆

None of 16 independent monoclonal antibodies against human (KB cell) DNA polymerase-alpha recognizes epitopes on cytoplasmic glucocorticoid receptors prepared from the same cells. Consistently negative results are obtained with separate assays that measure antibody binding to uncharged receptors or to charged receptor complexes that have been preloaded with a specific steroid ligand. These results must qualify the interpretation of possible immunological relations between polymerase-alpha and glucocorticoid receptors that were inferred from studies with polyclonal antisera of poorly defined specificity.