The DNA polymerases (gp43s encoded by gene 43
) of the related bacteriophages T4 and RB69 synthesize DNA with high fidelity, inserting one wrong nucleotide per 105
. These polymerases belong to the B family, the same family that is populated by the eukaryotic replicative polymerases α, δ, and ε. For the RB69 DNA polymerase, crystal structures are available for the apo enzyme2
, a binary editing complex with DNA bound in the exonuclease domain3
, and ternary complexes (replicating complex) with DNA and dNTP bound in the polymerase site4,5
. Because of the availability of crystal structures in so many conformations, the RB69 polymerase is a good structural model to study the mechanisms that are responsible for high base selectivity.
RB69 DNA polymerase achieves its fidelity by the coordinated action of two activities, a polymerase (Pol) activity that is responsible for correct nucleotide selection and an exonuclease (Exo) activity that removes mismatched nucleotides from the primer terminus. As with all polymerases that replicate DNA with high fidelity, binding the correct incoming dNTP introduces conformational changes in both the enzyme and the DNA. The fingers subdomain undergoes the largest conformational change, moving toward the palm subdomain so as to enclose the incoming dNTP and the complementary template nucleotide and thus to generate a binding pocket for the nascent base pair 4–8
In the RB69 polymerase, the active Pol binding site is formed by several highly conserved residues located in the fingers and palm. Arg482 and Lys560 in the fingers are involved in hydrogen bonding to the phosphate groups of the incoming dNTP. Hydrophobic interactions with the nascent base pair are formed by Leu415 and Tyr416 on the minor groove side and by Tyr567 and Gly568 on the template side. Asn564 and Ser565 form the rear wall of the binding pocket and ensure a coplanar base arrangement of the nascent base pair1,4,5,9
All of these residues play important roles in RB69 DNA replication fidelity. Leu415 and Tyr567 are critical for substrate discrimination. L415F/G mutants have elevated rates for a variety of base-substitution errors and single-base deletions in repetitive as well as nonrepetitive sequences9
. Replacing Tyr567 by Ala, Ser, or Thr confers a strong mutator phenotype, particularly for base-substitution errors1
. Tyr416 is responsible for discriminating between ribose and deoxyribose moieties in incoming nucleotides10
, while Leu561 discriminates sterically against mismatches in the nascent base-pair binding pocket11
. Thus, the size and shape of the polymerase binding pocket plays a crucial role in replication fidelity.
Insertion of a wrong nucleotide compromises the rate of primer extension. The polymerase stalls, altering the balance between extension by the polymerase and excision by the mismatch-editing exonuclease. Newly incorporated mismatches reduce the efficiency of subsequent nucleotide insertions and extensions by 102
- to 106
, the magnitude depending on the mismatch and the polymerase.
Polymerases with compromised fidelity due to alterations at the Pol site may display reduced partitioning to the Exo site and thus favor mismatch extension over proofreading, notably with the T4 PolL412M
and RB69 PolL415F/G
. The same mechanism probably applies to the RB69 PolY567A
mutant, as previously proposed1
. Conversely, T4 polymerase mutants such as T4A737V
that display an antimutator phenotype, mainly reducing A·T → G·C rates, tend to display decreased abilities to translocate after nucleotide incorporation, thus favoring the formation of exonuclease complexes and increasing the efficacy of proofreading15,16
. In preliminary screens, we observed that the RB69 S565G replacement had only weak impacts on fidelity but, surprisingly, when the Y567A and S565G replacements were combined within the highly conserved B motif KX3
G, the powerful mutator activity of the PolY567A
mutation was strongly reduced. To better understand the role of Ser565 in fidelity, we characterized some of the biochemical properties of RB69 polymerase mutants containing S565G, Y567A, or both replacements. We also investigated the impact of these replacements using fidelity assays both in vivo
and in vitro
. Our results lead us to propose that introducing the S565G replacement lowers the DNA binding affinity of the polymerase and increases its ability to dissociate from the primer-template, thus increasing the opportunity for proofreading by the same or another polymerase molecule (proofreading in trans
) and contributing to the antimutagenic impact of S565G on Y567A.