An attempt to unify the structure of polymerases

Abstract

With the great availability of sequences from RNA- and DNA-dependent RNA and DNA polymerases, it has become possible to delineate a few highly conserved regions for various polymerase types. In this work a DNA polymerase sequence from bacteriophage SPO2 was found to be homologous to the polymerase domain of the Klenow fragment of polymerase I from Escherichia coli, which is known to be closely related to those from Staphylococcus pneumoniae, Thermus aquaticus and bacteriophages T7 and T5. The alignment of the SPO2 polymerase with the other five sequences considerably narrowed the conserved motifs in these proteins. Three of the motifs matched reasonably all the conserved motifs of another DNA polymerase type, characterized by human polymerase alpha. It is also possible to find these three motifs in monomeric DNA-dependent RNA polymerases and two of them in DNA polymerase beta and DNA terminal transferases. These latter two motifs also matched two of the four motifs recently identified in 84 RNA-dependent polymerases. From the known tertiary architecture of the Klenow fragment of E. coli pol I, a spatial arrangement can be implied for these motifs. In addition, numerous biochemical experiments suggesting a role for the motifs in a common function (dNTP binding) also support these inferences. This speculative hypothesis, attempting to unify polymerase structure at least locally, if not globally, under the pol I fold, should provide a useful model to direct mutagenesis experiments to probe template and substrate specificity in polymerases.

Mutational analysis of two conserved sequence motifs in HIV‐1 reverse transcriptase

“…The same is true for the neighbourin8 mutation of Ala ~ ~ to Set (Table I) these mutated enzymes show reduced sensilivi[y to the inhibitors tested, which is particularly marked for the inhibition of the RT mutant (Asp=,Gly 'w~) by AZT-TP and ddTTP. The Tyr at position 115 is moderately well conserved [6,7] and its mutation to Asn results in a i0-fold increase in the Km for dTTP with a resulting drop In RT activity (Table 1), sugges|ing a degree of disruption at the active site. This mutant also displayed raised Kt's for AZT-TP and ddTTP and tile kinetics with PFA were non-linear, giving incomplete inhibition even at high PFA concentrations (only 60% inhibition was achieved at i.5 mM PFA), However, when the more conservative mutation to Phe was made, tl~e resulting protein had wild-type activity and Km for dTTP.…”

“…Various structure predictions, based on the known tertiary structure of the Klenow fragment of Pol I [14]. have placed these Asp's at the bottom of a cleft [7,15], but their precise location must await the solving of the X-ray crystallographic structure for HIV.I reverse transcriptase.…”

Complete sequence of RNA1 and subgenomic RNA3 of Atlantic halibut nodavirus (AHNV)

“…1). In all RdRps, 3 motifs are completely conserved including a GDD-consensus sequence which is believed to define a highly conserved 'polymerase site' (Poch et al 1989, Delarue et al 1990). The RdRp signature sequence has been defined as DX 3 (FYWLCA)X 0-1 DX n (STM)GX 3 TX 3 (NE)X n (GS)DD (Koonin & Dolja 1993), where X denotes any residue, residues in parentheses are amino acids allowed in this position, and subscripts denote the number of residues present.…”

Review

“…Structural elements of the palm and fingers subdomains of HIV-1 RT were found to form a clamp-like structure that holds the template-primer in precise orientation relative to the polymerase active site. These elements were named 'primer grip' and 'template grip' (JacoboMolina et al, 1993), and include sequences from conserved motifs E and B, respectively (Poch et al, 1989;Delarue et al, 1990). Most of the contacts between enzyme and template-primer involve the sugar-phosphate backbone of the DMA and are therefore not sequence specific.…”

“…These residues, which are well conserved within the pol genes of many retroviruses (motifs A and C, Larder et al, 1987b;Poch et al, 1989;Delarue et al, 1990), are involved in forming the topology of the nucleotide-binding site, which is located in the palm subdomain of the p66 subunit of HIV-1 RT. The most conserved element of this region in the HIV-1 RT and other lentiviruses have the YMDD motif (Tyr-Met-Asp-Asp amino acid residues 183-186) Jacobo-Molina et al, 1993).…”