T5 DNA polymerase: structural--functional relationships to other DNA polymerases.

Abstract

T5 DNA polymerase, a highly processive single-polypeptide enzyme, has been analyzed for its primary structural features. The amino acid sequence of T5 DNA polymerase has a high degree of homology with that of DNA polymerase I from Escherichia coli and retains many of the amino acid residues that have been implicated in the 3' -* 5' exonuclease and DNA polymerase activities of that enzyme. Alignment with sequences of polymerase I and T7 DNA polymerase was used to identify regions possibly involved in the high processivity of this enzyme. Further, amino acid sequence comparisons of T5 DNA polymerase with a large group of DNA polymerases previously shown to exhibit little similarity to polymerase I indicate certain sequence segments are shared among distantly related DNA polymerases. These shared regions have been implicated in the 3' -5' exonuclease function of polymerase I, which suggests that the proofreading domains of all these enzymes may be evolutionarily related.Bacteriophage T5 produces its own DNA polymerase that is essential for phage DNA replication (1). This DNA polymerase is unusual in that it is highly processive; it extensively elongates a primer before disassociating from the primer template and is capable of strand displacement. T5 DNA polymerase is more processive than any other singlepolypeptide DNA polymerase on comparable templates (2, 3). Similarly, its 3' -* 5' exonuclease or proofreading activity will processively hydrolyze hundreds of nucleotides before disassociation using either double-or single-stranded DNA substrates (4). The structural characteristics that confer high processivity upon polymerases are currently not understood, perhaps because other well-studied DNA polymerases require additional proteins to become processive. In contrast, processivity is an intrinsic property of T5 DNA polymerase and therefore it is an appropriate subject for investigation in the area of DNA replication.Furthermore, this polymerase is also rare in its ability to utilize nicked circular duplex DNA as a template and can unwind the parental DNA strand from its template as it synthesizes the new DNA strand from the 3'-OH end of the nick. The only other DNA polymerase capable of using a nicked template or of strand displacement in the absence of other protein factors is Escherichia coli DNA polymerase I (Pol I) or its large (Klenow) proteolysis product (4, 5). The dual properties of high processivity and strand displacement may make T5 DNA polymerase well suited for use in dideoxynucleotide DNA sequencing. The high processivity of modified T7 DNA polymerase-thioredoxin complex, known commercially as Sequenase, has made it very popular for use in DNA sequencing projects. Additionally, the strand-displacement ability of T5 DNA polymerase may enable it to proceed through double-stranded regions in template secondary structures or supercoiled plasmid templates.We present here the DNA sequence of the T5 DNA polymerase gene* and the deduced amino acid sequence of its product. Comparisons of the primar...

Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein

“…On the whole, 74% of the 640 codons of the oad gene end with A or T residues, with the highest preference (46.1%) for codons ending with T residues. The same has been observed with two other T5 genes, the D15 gene encoding 5' exonuclease (28) and the D7-8-9 gene encoding DNA polymerase (36); codons ending with T residues occur with a frequency of 43.6 and 44.9%, respectively.…”

Regulation of the pAD1-encoded sex pheromone response in Enterococcus faecalis: nucleotide sequence analysis of traA

“…The deduced protein of traA exhibited weak homology with several DNA-binding proteins: Bacillus subtilis BsuRI restriction enzyme (17% conserved plus 18% identical residues [30] within the first 281 residues of TraA), B. subtilis DNA polymerase III (15% conserved plus 15% identical residues [39]), and T5 DNA polymerase (14% conserved plus 19% identical residues [31] within the first 227 residues of TraA). The homology detected to the latter two proteins was within regions of these proteins predicted to play a role in 3'-*5' exonuclease activity.…”

Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

“…This motif must have arisen from a common ancestor sequence which evolved before the Gram-positive and Gramnegative bacteria diverged some 1.2 billion years ago [17]. We suggest that this motif is equivalent to the conserved YxxxD motif found among family A and B DNA polymerases [15,[18][19][20]. Recently, Brown and coworkers have also identified this HxAxxD motif and used site-directed mutagenesis on His 565 and Asp 570 in B. subtilis pol III to show the motif is critical for 3' to 5' exonuclease activity [21].…”