The long range objectives of this project are to characterize fundamental properties of the replication-specific mammalian DNA polymerase alpha (pol alpha) and its structural gene and to discern within them unique sites and/or mechanisms which can be exploited to develop novel pharmacologic agents with selective and useful anti-proliferative action.
Specific aims. This project proposes two approaches. One exploits DNA cloning and related techniques to isolate and study the nature of the gene(s) specifying pol alpha. The other involves the characterization of the structure and function of the enzyme, using novel pol alpha-selective, active site-directed purine nucleotide inhibitor/probes developed in this laboratory. These approaches are sorted into the following specific aims.
Aim I. To clone the gene(s) encoding the catalytic core of human pol alpha and to analyze their number, structure and transcription. Approaches include (a) identification and isolation of cloned pol alpha-specific gene sequences; (b) exploitation of the cloned sequences to isolate and characterize pol alpha-specific human genomic sequences and its/their transcription; and (c) attempts to produce full length pol alpha-specific cDNAs suitable for use in construction of pol alpha expression vectors.
Aim II. To continue characterization of the molecular basis of the inhibitory action and the pol alpha specificity of the butylphenylpurine dNTPs. The general approach will examine in detail how these agents work and simultaneously exploit them as specific probes to discern fundamental properties of the active site domain where they appear to act. The specific approaches include the use of: (i) procedures to study the effects of inhibitor forms on DNA:enzyme interactions; (ii) radiolabeled derivatives; (iii) irreversible cross-linking agents developed in Aim II, below; and (iv) physical and functional analyses of the inhibitor-specific binding site(s).
Aim III. To continue development of the pol alpha-specific active site-directed butylphenylpurine dNTP analogs - in particular substituted derivatives useful as pol alpha-specific marker molecules and as ligands for application in affinity-based identification and selective purification of pol alphas.
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