The goals of this work are to understand the enzymology and functions of human DNA polymerase delta.
Aim 1. The primary structures of human DNA polymerases delta-125 and delta-170 will be determined by cDNA cloning. The cDNAs for human DNA polymerase delta-125 and delta-170 will be cloned by a dual approach of hybridization screening of cDNA libraries and PCR amplification methods. Their nucleotide sequences and their encoded protein sequences will be determined. Final identification of the cDNA clones will be made by obtaining protein sequence data from the isolated proteins. This work will provide significant information on the structures of these two enzymes and permit an evaluation of their structural and evolutionary relationships with other members of this DNA polymerase family.
Aim 2. The chromosomal localization of the human DNA polymerase delta-125 and delta-170 genes will be determined by in situ hybridization. The organization of the DNA polymerase delta-125 gene will be investigated by Southern blot analysis and S1 nuclease mapping. The sequence of the 5' flanking region will be determined by the isolation of genomic clones from an EMBL3 genomic library using probes from the 5' end of the cDNA in order to obtain information on potential promoter sequences.
Aim 3. The expression of DNA polymerase delta activity will be examined by determination of the change in levels of enzyme activity, enzyme protein and steady state mRNA levels as a function of the cell cycle and proliferative state in human cultured cells. The potential promoter regions of the 5' flanking regions of the gene will be cloned into an expression vector containing a reporter gene and studied.
Aim 4. DNA polymerase delta-125 will be expressed in E. coli using an expression vector. The coding sequence will be inserted into the pET3a a vector for this purpose. The recombinant enzyme will be isolated, characterized and its properties compared to those of the native protein. Deletion mutants of the recombinant enzyme will be examined to obtain information on the potential domain structure of the enzyme. This work will provide significant information on the structures of these two enzymes and permit an evaluation of their structural and evolutionary relationships with other members of this DNA polymerase family and can contribute significantly to our understanding of mammalian replication. Studies of the human enzyme is of particular significance as it relates to the potential of basic studies to contribute to clinical aspects of health care. Basic information on a human proof-reading polymerase could contribute to our understanding of mechanisms of eukaryotic mutagenesis. Moreover, the genetic structures of these human enzymes are also of relevance to current efforts to sequence the human genome.
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