We propose to examine several aspects of DNA synthesis over the S-phase of highly-synchronous wild-type HeLa S3 cells. We will attempt to elucidate the nature and functional role of the three DNA polymerase alpha-containing replicative enzyme complexes, both as bound to, and as dissociated from, the nuclear matrix. The matrix-bound and cellular levels of DNA polymerase alpha, DNA primase, Topoisomerases I and II, a helix-destabilizing protein, dsDNA-dependent ATPase, RNase H, DNA ligases I and II, and ssDNA-binding proteins will be quantitated over the S- phase and over the G1-phase. The effects of the removal of total DNA and destabilized DNA on the binding of these enzymes and replicative complexes to nuclear matrices will be assessed. Dexamethasone-inducible cell constructs will be made by transfection with DNA constructs comprising linked svNEO, MMTV promoter, and c-myc sequences. HeLa constructs will be used to assess the effects of increased levels of nuclear oncogene products on DNA replication in a well-characterized, highly synchronous system. In addition, NIH 3T3 cell constructs will be used as a model of normal vs transformed cells. To determine whether changes in transcription levels over the cell cycle are responsible for changes in the levels of DNA polymerase alpha and beta enzymes, and of a helix-destabilizing protein, cDNA probes will be used in northern hybridization analysis. Finally, we will ascertain whether changing levels of replicative enzymes or of enzyme complexes over the S-phase correlate with changing chain elongation rates, or quantitative rates of DNA synthesis, or both.
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