Vibriophage phiVC8 DpoZ
ContentsIntroductionVibriophage phiVC8 DpoZ is a DNA polymerase belonging to the PolA family and the ΦVC8-like DpoZ subfamily, a group currently identified in certain species of bacteriophages[1]. DpoZ consists of two subfamilies: ΦVC8-like and Wayne-like. These polymerases confer selectivity in addition of the nucleobase 2-aminoadenine (Z) over adenine (A), with A completely ablated from their genomes. Z forms a non Watson-Crick base pair with thymine (T) consisting of three hydrogen bonds as opposed to the two present in A-T base pairing. Z is a relatively novel discovery, having only recently had its biosynthetic pathway described in detail[2]. DNA modifications in bacteriophages usually confer selective advantages by allowing phages to avoid host cell restriction enzyme digestion of their genomes[3]. The phage S-2L has a Z genome and encodes a PrimPol polymerase as well as a nucleotide phosphohydrolase called DatZ that removes the triphosphate tail of only dATP, excluding it from the genome[4]. Vibriophage ΦVC8 also encodes DatZ[5], though it is unknown if it is the primary enzyme responsible for A exclusion or if its polymerase also plays a role. The specific mechanisms by which ΦVC8-like DpoZ polymerases carry out A exclusion and Z inclusion are still under investigation, though specific structural features and putative specificity mechanisms are highlighted below. Structural HighlightsThe 2.8Å crystal structure solved of the 646 amino acid protein contains two domains: a polymerase domain and a  The structure deposited in PDB (7pbk) has two conformations: thumb-exo open and thumb-exo closed. These conformations involve movement of the thumb and exonuclease domains. ΦVC8 DpoZ closely resembles E. coli DNA Polymerase I Klenow fragment, containing distinct palm, thumb, and fingers subdomains (palm in blue, thumb in green, fingers in pink, subdomains approximated from related polymerases). The enzyme exhibits the typical fold of PolA polymerases including E. coli Klenow fragment and T7 DNA polymerase. The residues K162 and G276 appear to have the largest positional shifts between the two conformations. The palm subdomain contains the polymerase active site, where the thumb and fingers clamp onto a DNA substrate to hold it in place. The 3'-5' exonuclease site has a shift in residues R161 and M165 from the open conformation and the closed conformation.  When aligned with other prokaryotic PolA polymerases[7], three unique insertions in the polymerase domain are present. The first ranges from the residues 349-353 in the palm subdomain. The second is adjacent to helix O in the fingers subdomain from residues 442-448, with this insertion disrupting a part of motif B containing helix O. The third is between helices O1 and P on the tip of the fingers, from residues 473-491. The longest of these insertions (3) is very flexible, not being well defined on the electron density map, indicating that DNA binding may be necessary for stabilization of that loop. The first and third insertions likely interact with dsDNA, though solving a ternary complex structure is required to confirm this. The second insertion (residues 442-448) adds a loop in the helix structure that normally interacts with dNTPs[8]. FunctionPolymerase domainThe polymerase active site contains 10 residues essential to catalyzing nucleotide addition to the template strand. The universally conserved catalytic dyad (H581 and D582) is present and key residue R440, responsible for stabilizing the gamma phosphate of incoming dNTPs, is also conserved. Still, a number of residues in the polymerase domain differ from other PolA polymerases. L455, F459, G548, and S583 are all conserved mutations in the ΦVC8 DpoZ subfamily, though not in the Wayne-like DpoZ subfamily[9]. L455 and G548 do not appear in any known Wayne-like DpoZ subfamily structures, though the F459 residue is present. F459 is normally a tyrosine residue that acts as a steric gate for distinguishing dNTPs from NTPs[10], though this function may be conserved as was shown for T. aquaticus DNA polymerase with a Tyr to Phe mutation in a previous study[11]. In any case, these individual changes from other polymerases may help account for specificity in Z nucleobase recognition, as it is likely that it is not a single mutation in the enzyme that accounts for the selectivity. Exonuclease domainThe exonuclease domain of ΦVC8 DpoZ is larger and contains two non-canonical helices when compared with other PolA family members. These helices are referred to as E1 and E2. This domain also contains an insertion between helices a4 and a5 present in other phages, such as T7, and is implicated in shuttling the 3' end of the DNA primer between the polymerase and exonuclease sites[12]. The primary feature of ΦVC8 DpoZ and other DpoZ family members is its selectivity in Z vs A incorporation. The exact mechanism by which this occurs is still under investigation and cannot be definitively described, though there is a proposed mechanism based off of the currently solved structure (7pbk). The noncanonical helices E1 and E2 comprise an unusually mobile portion of the exonuclease domain that may contribute to proofreading properties. Residues R161 and M165 of helix E2 have been shown to contact the base of leaving nucleotides when modeled. Mutating these residues in a double mutation to alanine resulted in a detrimental effect in exonuclease activity and about equivalent efficiencies of Z and A incorporation, though interestingly also resulted in a higher incorporation of Z over A that has not been investigated or accounted for. These residues may still have an important function in Z vs A incorporation that requires further investigation. 2-aminoadenine IncorporationSelectivity of Z vs A may result from the enzyme exhibiting selectivity in incorporating base pairs with three hydrogen bonds and excluding those with two (A-T). This could occur by polymerase backtracking, a feature previously thought to be distinct to RNA-dependent DNA polymerases but has also been shown to occur in some DNA-dependent DNA polymerases[13]. Polymerase backtracking occurs when the enzyme disengages from the nascent 3' end of DNA and moves backwards on the template strand. This proposed mechanism may allow the enzyme to be more sensitive to base-pairing interactions and differentiate between two vs three hydrogen bonds in a base pair. This ability would be enhanced in the enzyme compared to other polymerases and the energy barrier between backtracking conformation and forward moving polymerization would need be to significantly lowered. This may work through an allosteric mechanism that gradually allows the transition of the enzyme from the polymerase domain to the exonuclease domain to excise any bases with fewer than three hydrogen bonds. The insertion at residues 117-127 of the exonuclease domain may be involved in such a mechanism, though this is yet to be tested and would require analysis of a ternary complex. Further investigation into the mechanisms behind Z incorporation and selectivity is ongoing and may soon shed more light on the specific structural feature necessary to facilitate such specificity.
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