We continued biochemical studies of mammalian DNA replication proteins. cDNAs for rat and human beta-polymerase have been subcloned in expression vectors, and the proteins have been overproduced in E. coli, purified in mg quantities, and sequenced. Structure-function aspects and in vitro DNA repair activities of the recombinant enzymes are being studied. Genomic DNA spanning the gene for human beta-polymerase was further characterized. The promoter region was studied by detailed deletion and site-directed mutagenesis using a transient expression system for assay. In other work, we purified and characterized a transcription factor for the human beta-polymerase gene. In vitro transcription studies of the gene are under way, as well as physical biochemical studies of the sequence-specific binding between the promoter and the purified factor. Studies of cell lines carrying inducible beta-polymerase mRNA antisense transcripts revealed that down regulation of beta-polymerase expression is associated with a block in DNA repair after nitrogen mustard treatment of cells.
