2-Chloro-2'-deoxyadenosine, a nucleoside analog with potential efficacy in the treatment of lymphoid neoplasias, is under active investigation in Phase II pediatric and adult clinical trials. The compound's ultimate clinical value will depend on a more complete understanding of its mechanism(s) of action, its mutagenicity, and its repairability upon incorporation into DNA, which could be a factor in therapeutic efficacy. Hence, the overall goal of this project is to determine the functional consequences of incorporation of the analog's triphosphate form, CldATP, into DNA. This event is postulated to interfere with subsequent DNA synthesis and RNA transcription, to alter the fidelity of DNA polymerization, and to be recognized as an abnormal base by proteins involved in DNA repair. These predictions will be tested in four series of experiments designed with the following aims:
Specific Aim 1. Determine the effects of CldATP incorporation on subsequent DNA polymerization by examining the susceptibility of ClA-containing nascent DNA strands to 3'-5' exonucleolytic degradation, the efficiency of bond formation between ClA residues and adjacent nucleotides, and the ability of DNA-containing ClA residues to serve as a template for in vitro DNA synthesis.
Specific Aim 2. Determine the fidelity of DNA polymerization in the presence of ClA by site-directed mutagenesis and the phiX174am16 assay that detects single base substitutions within an amber mutation, by use of the M13mp2 forward mutation assay that detects changes at many sites within a nonessential gene, and by the analysis of mammalian cell mutation induction within the human hgprt locus and in a transfected phage shuttle vector.
Specific Aim 3. Determine the effects of ClA-substituted DNA on RNA transcription by examining promoter function, the extent and rate of elongation of RNA transcripts, and fidelity of base pairing during in vitro transcription of ClA-containing DNA.
Specific Aim 4. Identify DNA-binding proteins or DNA repair enzymes specific for ClA residues by first assaying for recognition of this abnormal base in DNA by purified E. coli ABC excinuclease and, secondly, by examining human cell extracts in a gel shift binding assay to identify novel DNA binding proteins that bind to or repair ClA-containing DNA. The information gained from these studies will provide needed insights into the effects, both short- and long-term, of a chlorinated purine in DNA. In addition, it may be possible from the mutagenicity data to predict the analog's carcinogenic potential or lack thereof.
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