A 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.

The bacteriophage PRD1 is a lipid bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they harbor the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. Like adenovirus and the Bacillus phage φ29, PRD1 specifies its own DNA polymerase which is able to utilize the phage encoded terminal protein to prime DNA synthesis. In addition to these two proteins, PRD1 also specifies an additional replication protein (p12) of unknown function. We have sequenced the origins of replication (termini of the genome) as well as the right most 1700 bp of the bacteriophage PRD1 genome. The right most 1700 bp encompasses the right early region and completes the sequence of all PRD1 early functions. We report here that the PRD1 genome contains a perfect 111 bp inverted terminal repeat. Furthermore, statistical analyses of the right 1700 bp, as well as the examination of transcription and translation signals has allowed us to assign gene XII to an open reading frame and to infer the direction of both early and late transcription. Gene XII, which has been implicated in the replication process and the regulation of gene expression is predicted to encode a 16.7 Kdal protein. Data base searches have revealed a possible evolutionary relationship between this protein and the ε-subunit of E. coli DNA polymerase III. We have also mapped right, early transcription of the PRD1 genome. This has corroborated our inference concerning the direction of right early transcription and confirmed our assignment of gene XII to an open reading frame. It has also revealed that two putative rho-independent terminators are functional in vitro and that the putative right early promoter is utilized in vivo and in vitro. The data presented here have permitted us to ascertain the general genomic and transcriptional organization of PRD1 and to predict the primary structure of the product of gene XII. These results, in turn, have allowed us to develop hypotheses concerning the evolution of linear, protein primed DNA's and the function of gene XII.