An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3'-5' proof-reading exonuclease activity is also necessary.

DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction.

The polymerase chain reaction (PCR) is widely applied across the biosciences, with archaeal Family-B DNA polymerases being preferred, due to their high thermostability and fidelity. The enzyme from Pyrococcus furiosus (Pfu-Pol) is more frequently used than the similar protein from Thermococcus kodakarensis (Tkod-Pol), despite the latter having better PCR performance. Here the two polymerases have been comprehensively compared, confirming that Tkod-Pol: (1) extends primer-templates more rapidly; (2) has higher processivity; (3) demonstrates superior performance in normal and real time PCR.

Human immunodeficiency virus reverse transcriptase displays dramatically higher fidelity under physiological magnesium conditions in vitro.

Unlabelled: The fidelity of human immunodeficiency virus (HIV) reverse transcriptase (RT) has been a subject of intensive investigation. The mutation frequencies for the purified enzyme in vitro vary widely but are typically in the 10(-4) range (per nucleotide addition), making the enzyme severalfold less accurate than most polymerases, including other RTs. This has often been cited as a factor in HIV's accelerated generation of genetic diversity.

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3'-5' proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny.

A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement.

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor.