Structure-function analysis of PRD1 DNA polymerase; nucleotide sequence, overexpression and in vitro mutagenesis of the PRD1 DNA polymerase gene.
KeywordsDNA polymerases -- Analysis.
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PublisherThe University of Arizona.
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AbstractA small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase which utilizes terminal protein as a primer for DNA synthesis. The PRD1 DNA polymerase gene has been sequenced and its amino acid sequence deduced. This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300. Thus, it is the smallest DNA polymerase ever isolated from prokaryotic cells. Comparison of the PRD1 DNA polymerase with other DNA polymerases whose sequences have been published, yielded segmental but significant homologies. These results strongly suggest that many prokaryotic and eukaryotic DNA polymerase genes regardless of size have evolved from a common ancestral gene. The results further indicate that those DNA polymerases which use either an RNA or protein primer are related. We propose to classify DNA polymerases on the basis of their evolutionary relatedness. In order to overexpress PRD1 DNA polymerase in E. coli cells, the 2kb Hae II fragment was isolated from phage genomic DNA. This fragment was then cloned into pEMBLex3 expression vector. Phagemid pEMBLex3 contains lambda pR promoter and cI857 gene as a repressor. A specific 57 bp deletion was performed by using uracil containing ss DNA and oligonucleotide spanning each region to remove an unwanted non-coding region. After this deletion, the PRD1 DNA polymerase gene is totally under the control of the vector promoter and SD sequence. Upon heat induction, a protein with an apparent size of 68 kdal was overexpressed as an active PRD1 DNA polymerase. The expression of DNA polymerase was about 1% of total E. coli protein. The PRD1 DNA polymerase is a small multifunctional DNA polymerase and has three major conserved amino acid sequences which are shared among many DNA polymerases including human DNA polymerase alpha. Therefore, the PRD1 DNA polymerase provides an useful model system to study structure-function analysis of DNA polymerases. Four specific amino acid changes generated in conserved regions by the site-directed mutagenesis, in order to investigate their functional roles. Based on complementation test, three conserved regions are functional domains of PRD1 DNA polymerase.
Degree ProgramMicrobiology and Immunology