An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.
Bottom Line: The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers.The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs.Tgo-Pol Z1 may also be useful for amplification of damaged DNA.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK firstname.lastname@example.org.Show MeSH
Related in: MedlinePlus
Mentions: Pyrococcus furiosus (Pfu), and the closely related Thermococcus gorgonarius (Tgo), are hyperthermophiles from the euryarchaeal order Thermococcales (32), which furnishes most of the polymerases used in PCR applications that require high accuracy (23–25). Mutations to an aspartic acid, located in a three amino acid loop linking the two long α-helices that make up the fingers domain (Figure 1), decrease the accuracy of Pyrococcus furiosus Pol B (Pfu-Pol) (33). In both Pfu-Pol and Tgo-Pol, as previously documented (33), the critical aspartic acid makes a network of hydrogen bonds to a set of amino acids located in the following helix (Figure 1, Table 1). No structure exists for the most thoroughly investigated mutant, Pfu-Pol D473G (33), but a ‘Phyre2’ threading model suggests a loop extended by one amino acid with retention of only a single hydrogen bond (Figure 1, Table 1). The aspartic acid uses both side chain and peptide bond atoms for hydrogen bonding, explaining the persistence of an interaction with D473G. The accurate eukaryotic Pols α and δ have an almost identical fingers domain to the archaeal polymerases; amino acid sequence and structural comparisons both show a near superimposable three amino acid loop with retention of the aspartic acid and similar hydrogen bonding to downstream amino acids (Figure 1, Table 1). Sequence alignments suggest that Pol ζ has a longer loop and, although structural information is lacking, modelling is also consistent with elongation (Figure 1, Table 1). Two hydrogen bonds result, between D1070 (the D473 equivalent) and amino acids +3 (T1073) and +5 (K1075) along. A further manipulation to the fingers loop, commencing with a proof-reading exonuclease variant of Tgo-Pol, replaced the natural three amino acids with the longer sequence found in Sce-Pol ζ (Figure 1, Table 1). The resulting derivative, referred to as Tgo-Pol Z1, possessed reverse transcriptase activity and had low fidelity (34). It is probable that Tgo-Pol Z1 has an expanded flexible active site able to accommodate the non-B DNA structures of DNA–RNA heteroduplexes (35) formed when RNA is copied with dNTPs. Again, no structures have been determined for Tgo-Pol Z1 but a model is consistent with that of Sce-Pol ζ, i.e. a long loop with hydrogen bonds between the aspartate and succeeding residues (Figure 1, Table 1). The sequences and structures of the fingers domains of Saccharomyces cerevisiae polymerase epsilon (Sce-Pol ϵ) and bacterial Eco-Pol II are also shown in Figure 1. Although a Sce-Pol ϵ replicase possesses an extended loop and demonstrates only a single hydrogen bond between D807 (equivalent to D473) and downstream amino acids (Table 1). In contrast, although Eco-Pol II belongs in the translesion category, the fingers loop is short (three amino acids) and N485 (the spatial counterpart of D473) makes three hydrogen bonds with the succeeding α-helix. Some observations in Figure 1 and Table 1 are based on modelled rather than real structures and merit a degree of caution.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK email@example.com.