Learning from Directed Evolution: Thermus aquaticus DNA Polymerase Mutants with Translesion Synthesis Activity

Abstract

DNA is being constantly damaged by endo- and exogenous agents such as reactive oxygen species, chemicals, radioactivity, and ultraviolet radiation. Additionally, DNA is inherently labile, and this can result in, for example, the spontaneous hydrolysis of the glycosidic bond that connects the sugar and the nucleobase moieties in DNA; this results in abasic sites. It has long been obscure how cells achieve DNA synthesis past these lesions, and only recently has it been discovered that several specialized DNA polymerases are involved in translesion synthesis. The underlying mechanisms that render one DNA polymerase competent in translesion synthesis while another DNA polymerase fails are still indistinct. Recently two variants of Taq DNA polymerase that exhibited higher lesion bypass ability than the wild-type enzyme were identified by directed-evolution approaches. Strikingly, in both approaches it was independently found that substitution of a single nonpolar amino acid side chain by a cationic side chain increases the capability of translesion synthesis. Here, we combined both mutations in a single enzyme. We found that the KlenTaq DNA polymerase that bore both mutations superseded the wild-type as well as the respective single mutants in translesion-bypass proficiency. Further insights in the molecular basis of the detected gain of translesion-synthesis function were obtained by structural studies of DNA polymerase variants caught in processing canonical and damaged substrates. We found that increased positive charge of the surface potential in the area proximal to the negatively charged substrates promotes translesion synthesis by KlenTaq DNA polymerase, an enzyme that has very limited naturally evolved capability to perform translesion synthesis. Since expanded positively charged surface potential areas are also found in naturally evolved translesion DNA polymerases, our results underscore the impact of charge on the proficiency of naturally evolved translesion DNA polymerases.

Amino Acid Templating Mechanisms in Selection of Nucleotides Opposite Abasic Sites by a Family A DNA Polymerase

“…Abasic Site-All KlenTaq crystals grew in the same space group and very similar cell parameters (supplemental Table S1) as those reported earlier (19,20,35,(42)(43)(44)(45). Thus, significant differences in crystal packing forces acting on the active site should be negligible.…”

“…Structure of KlenTaq in Binary Complex with DNA Duplex Containing an Abasic Site (KlenTaq F-binary )-To address the issue, if the overall conformation of the enzyme-DNA complex is affected by an abasic site prior to nucleotide binding, we crystallized KlenTaq bound to primer/template duplex containing an abasic site following a similar strategy as reported recently (19,20,35,43,46). The structure was solved by difference Fourier techniques at a resolution of 1.9 Å (supplemental Table S1).…”

“…S1) have shown that purines, in particular adenosine, and to a lesser extent guanosine, are most frequently incorporated opposite the lesion. The strong preference for adenosine has been termed "A-rule" (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

“…Superior stacking as well as solvation properties of adenine have been discussed as the driving force behind adenine selection (17, 29 -31). However, previously reported structures of KlenTaq, the large fragment of Thermus aquaticus (Taq) DNA polymerase I, which is a member of the sequence family A, suggests that this enzyme follows the A-rule by applying an amino acid templating mechanism (19,20). Thereby, interaction with Tyr-671 seems to be the basis of the preference of purines.…”

Morphology, Physiological Characteristics, and Complete Sequence of Marine Bacteriophage ϕRIO-1 Infecting Pseudoalteromonas marina

“…To align among groups, each group alignment was converted to an hhpred-style hidden Markov model, and model-to-model alignment was carried out using a local implementation of the hhsearch software (31). The bacterial polymerase with Protein Data Bank (PDB) accession number 3PO4 (32) and its relatives were found to have the minimal structure. Each group of sequences was first aligned to the sequences of the members of the family with PDB accession number 3PO4 using hhsearch.…”