Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Abstract

All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3′-5′ exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.

Bacteriophage-Encoded DNA Polymerases—Beyond the Traditional View of Polymerase Activities

“…DpoZ enzymes from four different phages, overproduced in E. coli and purified to homogeneity, functionally resemble the Klenow fragment of family A DNA polymerase I, but show significantly higher affinity to dZTP over dATP [ 64 ]. The crystal structure of the apo form of DpoZ from vibriophage ϕVC8 showed that this polymerase is structurally quite a typical member of the A family [ 65 ]. It consists of a polymerase domain with palm, thumb, and fingers subdomains as well as a 3′→5′ exonuclease domain.…”

“…Compared to E. coli Pol I, ϕVC8 DpoZ has three small sequence insertions that form external loops in the palm and fingers domains. However, neither single structural motif nor specific residue(s) in the polymerase domain that could justify the preference for dZTPs during DNA synthesis have been identified [ 65 ]. Therefore, substrate preference may arise from the combination of several structural features.…”

“…It was recently shown that in response to replication obstacles, DNA polymerase backtracks the nascent DNA strand and sends it to the exonuclease domain, where even a correctly incorporated nucleotide is excised [ 66 ] ( Figure 6 B). According to Czernecki and coworkers, backtracking activity of DpoZ enzyme could be especially high and somehow sensitive to the strength of the nascent base pair, that is to the number of forming hydrogen bonds, in favor of aminoadenine [ 65 ] ( Figure 6 C). Structural basics of this mechanism remain yet to be elucidated.…”

“…However, the nascent strand (grey fragment) is occasionally backtracked and in consequence DNA is uncoupled and single strand fragments become disordered (dotted lines). As a result, the primer strand is sent to the exonuclease site (polymerase in light purple), where 2–3 terminal nucleotides are subsequently removed before switching to the extending mode (adapted from [ 65 ]). ( C ) DpoZ specificity may relate to high backtracking activity dependent on the number of formed hydrogen bonds.…”

Mechanisms supporting aminoadenine-based viral DNA genomes

“…They also propose that excision of incorporated dA may be promoted by the 3′-exonuclease activity of DpoZ [27]. However, a close investigation of the exonuclease domain of PhiVC8 DpoZ suggests that the selectivity towards dZTP is not directly encoded in its active site [28].…”