The quest for optimal DNA sequencing reagents presents a formidable challenge given the variety of polymerases, nucleotide analogs and reaction conditions available. These studies identify three elements that enhance terminator incorporation, thus facilitating DNA sequence determination. These elements are (i) acyNTPs, a preferred substrate for archaeon DNA polymerases, (ii) dyes that enhance incorporation when conjugated to nucleoside bases and (iii) archaeon DNA polymerase variants with enhanced ability to incorporate chain terminators such as ddNTPs and acyNTPs. To a first approximation, these elements act in an additive way, with each element making a distinct contribution (Fig. ). Thus, the 8000-fold enhancement of ROX–acyCTP incorporation by Vent A488L DNA polymerase, compared with ddCTP incorporation by Vent DNA polymerase, can be broken down into its component parts: a 32-fold enhancement due to the ROX dye, 16-fold due to the acyCTP and 16-fold due to the A488L mutation.
Figure 6 Incorporation enhancement attributable to terminator and polymerase variations. Incorporation is represented on a logarithmic scale relative to ddCTP incorporation by Vent DNA polymerase. Values for ThermoSequenase incorporation of ddCTP are inferred (more ...)
Incorporation of acyNTPs by Family B DNA polymerases has previously been studied in the context of the antiviral activity of analogs such as acyclovir (i.e. acycloguanine). The Family B DNA polymerases from Herpes virus type 2 and human cytomegalovirus insert acyGTP more readily than ddGTP (21
), a characteristic shared with the hyperthermophilic archaeon DNA polymerases tested here. In contrast, human DNA polymerase α incorporates acyGTP less efficiently than ddGTP (21
). This brief survey of polymerases demonstrates that classification in Family B is insufficient to predict whether ddNTPs or acyNTPs will be more efficiently incorporated. Nonetheless, we anticipate that, given the high degree of sequence similarity, most, if not all, hyperthermophilic archaeon Family B DNA polymerases will share the terminator incorporation patterns we have observed. Sequence similarities suggest that other archaeon non-thermophilic DNA polymerases may also follow this pattern.
DNA polymerase discriminates against ddNTPs and even more strongly against acyNTPs (Fig. A). In the case of ddNTP, discrimination is manifest in a decreased rate of phosphodiester bond formation, as opposed to a decrease in the affinity of substrate binding (22
). The decreased rate has been ascribed to loss of hydrogen bonding between the 3′-OH of the substrate dNTP and the pro-Sp oxygen in the same dNTP (23
), possibly affecting alignment of the reactive groups. In contrast, the F667Y variant of Taq
DNA polymerase (Thermo Sequenase; 20
) modestly discriminates against ddNTPs and only slightly more against acyNTPs (Fig. C), presumably reflecting the ability of the substituted tyrosine hydroxyl group to substitute for the missing 3′-OH. Indeed, T7 DNA polymerase containing tyrosine at the analogous position does not distinguish between dNTP and ddNTP incorporation, a fact reflected in virtually identical kinetic parameters involving nucleotide binding and phosphodiester bond formation (22
While the activity of the F667Y Taq
variant can be rationalized in direct terms, the effects of DNA polymerase variants 9°N A485L, Vent A488L and Pfu
) are less apparent. The crystal structures of the apo 9°N DNA polymerase (24
) and of the Family B RB69 DNA polymerase replication complex (11
) predict that this residue will face away from the active site, with little likelihood of directly interacting with the nucleotide substrate. Thus, this mutation appears to operate indirectly, perhaps affecting the conformational shift noted prior to polymerization.
The RB69 replication complex exhibits stacking interactions between the ribose of the substrate TTP and Y416, an amino acid conserved in Family B archaeon DNA polymerases and implicated in ribonucleotide discrimination (12
). Local sequence and crystallographic similarities suggest that this stacking will also be available in the archaeon DNA polymerases. Even though such stacking cannot occur with acyclic analogs, such analogs are preferred substrates over dideoxynucleotides, perhaps due to the increased conformational flexibility inherent in the acyclic structure. It seems somewhat paradoxical that in the case of the T7 DNA replication complex no stacking interactions with the ribose ring are predicted and yet dideoxy are favored over acyclic analogs. Indeed, given the differing acyNTP and ddNTP sensitivities, it seems probable that alternate sugar contacts are involved in Family A and B polymerases, contacts that ultimately affect positioning of active site residues. Thus, despite global similarities, significant differences appear to exist in the active sites of these two families of DNA polymerases.
At present it is difficult to predict the structural or chemical basis by which dye substituents enhance nucleotide incorporation. Ilsey and Buzby noted in the series of compounds tested that the most hydrophobic indocyanine or rhodamine dyes were preferred substrates for polymerization (13
). However, a limited number of dyes were tested, making it difficult to predict which elements are most important in the observed incorporation bias. Presumably, the dye effect arises from enhanced binding of the base, although effects on the transition into an active polymerization complex cannot be excluded.
The apparent additivity of dye substitution, terminator type and mutations in facilitating terminator incorporation suggests that these effects act independently. In the absence of further kinetic studies it is not possible to state whether they act on the same rate limiting step or even if the same rate limiting step is encountered in terminator incorporation as opposed to dNTP incorporation. Nor have the effects of binding and the chemistry of bond formation been separately identified. More detailed kinetic and structural studies hold the promise of dissecting the components involved in the observed discrimination. Hopefully such studies will illuminate not only the basis of the observed effects, but also lend insight into the operation of the active site in DNA polymerization.
One outcome of these studies is an alternative environment for DNA sequencing, applicable in both long range primary sequence determination (Fig. ) and in the short extensions required for SNP analysis. Incorporation dynamics are comparable to dye–ddNTP incorporation by Taq F667Y DNA polymerase variants, allowing the use of low concentrations of dye–terminators while retaining sequence specificity. As such, these reagents are poised to complement and extend currently available methods of DNA sequence analysis.