A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.

Abstract

Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by >2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250-to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.All known DNA polymerases can be placed in one of four families based on sequence homologies (1, 2). Although Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase (Taq DNA polymerase), and bacteriophage T7 DNA polymerase are members of the DNA polymerase I (Pol I) family, a number of their properties differ significantly (3).First, T7 DNA polymerase is responsible for the replication of a genome and, as such, interacts with other replication proteins, whereas E. coli DNA polymerase I and Taq DNA polymerase are mainly responsible for repair and recombination. Second, the three polymerases have different exonuclease activities: T7 has only a 3' --5' activity, Taq has only a 5' --3' activity, and E. coli DNA polymerase I has both 3' --5' and 5' --3' activities. The exonuclease activities reside in separate domains at the amino termini and can be inactivated selectively by genetic modification. Third, the thermostability of Taq DNA polymerase distinguishes it from the other two. A fourth difference is their relative abilities to distinguish between a deoxy-and a dideoxyribose in the nucleoside triphosphate. E. coli DNA polymerase I (4-6) and Taq DNA polymerase (7) incorporate deoxyiucleotides at a rate that is several hundred to several thousand times that of dideoxynucleotides, while T7 DNA polymerase incorporates dideoxynucleotides much more efficiently, preferring deoxynucleotides by only a factor of three (5, 6). To determine the molecular basis for this difference we constructed active site hybrids of the DNA polymerases and examined their ability to use ddNTPs relative to dNTPs. Inasmuch as chain termination by dideoxynucleotides is the basis of all commonly used enzymatic methods of DNA sequencing (5, 8), an understanding of this difference has utility for improving the DNA polymerases used for this purpose.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6339METHODS Construction ofHybrid Genes. Hybrid genes were constructed by using synthetic oligonucleotides and...

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

“…Site-directed mutagenesis of Phe762 to tyrosine in DNA polymerase I from E. coli and Thermus aquaticus (Taq polymerase) increases the incorporation of ddNTPs 1000-fold or more than the native enzymes (Tabor and Richardson, 1995), a feature essential for DNA sequencing by chain-termination (Tabor and Richardson, 1987). Tyrosine occurs naturally at this position in Mycobacterium spp.…”

“…and some phage polymerases, including that from phage T7 (Tyr526) (Tabor and Richardson, 1987;Doublie et al, 1998). Despite a low dNTP:ddNTP incorporation ratio of three (Tabor and Richardson 1995), T7 polymerase contains a strong 3 0 -5 0 exonuclease capable of degrading ddNTPs (Tabor and Richardson, 1987). Thus, the selective pressure of tyrosine at this position in phage T7 is likely not the incorporation of ddNTPs but rather increased efficiency.…”

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine

“…One of the interesting properties of T7 DNA polymerase is its ability to incorporate dideoxynucleoside monophosphates (ddNMPs) more efficiently than most DNA polymerases, including the homologous E. coli DNA polymerase I (14) and Thermus aquaticus DNA polymerase (15). This increased ability is due to a single amino acid residue, tyrosine-526, in T7 DNA polymerase; both E. coli DNA polymerase I and Taq polymerase have a phenylalanine at the equivalent position (15).…”

“…This increased ability is due to a single amino acid residue, tyrosine-526, in T7 DNA polymerase; both E. coli DNA polymerase I and Taq polymerase have a phenylalanine at the equivalent position (15). The crystal structure of a T7 DNA polymerase-dNTP complex shows the hydroxyl group of the tyrosine interacting with the ␤-phosphate of the ddNTP, presumably stabilizing it (16).…”

Toward a Complete Human Genome Sequence

“…Changes in DNA sequencing chemistry have led to substantial improvement in the quality and read length of the primary sequence, with no extra labor investment per sample. These changes include the introduction of fluorescent energy transfer dyes (Smith et al 1985;Ju et al 1995;Rosenblum et al 1997) for more sensitive labeling of DNA and engineered thermostable polymerases (''FY'') that are more processive and less discriminant in incorporating dideoxy nucleotides (Tabor and Richardson 1995). A complementary approach has been to increase the number of samples loaded per run.…”