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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028775-07
Application #
3276066
Study Section
Biochemistry Study Section (BIO)
Project Start
1981-07-01
Project End
1991-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Barnes, M H; Hammond, R A; Kennedy, C C et al. (1992) Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III. Gene 111:43-9
Hammond, R A; Brown, N C (1992) Overproduction and purification of Bacillus subtilis DNA polymerase III. Protein Expr Purif 3:65-70
Khan, N N; Wright, G E; Brown, N C (1991) The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization. Nucleic Acids Res 19:1627-32
Hammond, R A; Barnes, M H; Mack, S L et al. (1991) Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene. Gene 98:29-36
Butler, M M; Dudycz, L W; Khan, N N et al. (1990) Development of novel inhibitor probes of DNA polymerase III based on dGTP analogs of the HPUra type: base, nucleoside and nucleotide derivatives of N2-(3,4-dichlorobenzyl)guanine. Nucleic Acids Res 18:7381-7
Wright, G E; Brown, N C (1990) Deoxyribonucleotide analogs as inhibitors and substrates of DNA polymerases. Pharmacol Ther 47:447-97
Barnes, M H; Hammond, R A; Foster, K A et al. (1989) The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III. Gene 85:177-86
Talanian, R V; Brown, N C; McKenna, C E et al. (1989) Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta. Biochemistry 28:8270-4
Hammond, R A; Foster, K A; Berchthold, M W et al. (1988) Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerase alpha. Biochim Biophys Acta 951:315-21
Foster, K; Luthi-Steinmann, K; Barnes, M et al. (1986) Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha. Biochem Biophys Res Commun 140:21-7

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