Abstract
Bacteriophage T4 gene 43 codes for the viral DNA polymerase. We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing. We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E. coli containing a gene 43 overexpression vector. A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase. All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572. Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha. Surprisingly, T4 DNA polymerase shares only limited similarity with E. coli polymerase I and no detectable similarity with T7 DNA polymerase. Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.
Highlights
The DNA was extracted with phenol/chloroform, of the T4 genome that contains the three genes encoding DNA polymerase accessory proteins and theregA gene, as shown in Fig. 1(Wood and Revel, 1976; Kutter andRuger, 1983).Wilson et al (1977) have previously constructeda X-T4 recombinant phage(X-NM806-17) that (l:l, (v/v)) and precipitated with ethanol
Since no other aminoassisted comparison of the sequence of T4 DNA polymerase acid changes were detected by comparison of HPLC tryptic with E. coli DNA polymerase I (Joyce et al, 1982) and peptide maps of the wild type and serine variant, the serine T 7 DNA polymerase (DunnandStudier, 1983) using the substitution at residue 214 appears to be responsible for the program SEARCH
We report here that threegions of homology with T 4 DNA polymerase are more extensive than previously noted andhomologous sequences are present in the DNA polymerase from human cytomegalovirus (HCMV) (Kouzarides et al, 1987), varicella zoster (VZV) (Davidson and Scott, 1986), and human DNA polymerase a (Wong et al, 1988)
Summary
Dept. of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114. DNA polymerases thanit does with those of procaryotic origin. Of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114. DNA polymerases thanit does with those of procaryotic origin. The location of specific mutations in herpes simplex virus DNA polymerase andT4 DNA polymerase andthe conservation of common amino acid sequences suggest that monton, Canada T6G 2E9. Medical University of South Carolina, Charleston, SC 29425. In T4 DNA polymerase the nucleotide-binding site is located Purification of T4DNA Polymerase"T4 DNA polymerase was in the COOH-terminal half of the protein. Purified from (a) E. coli BB cells infected with T4D g42amN55, g30amH39, regAsp62and ( b )induced cells containing the expression
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