Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene

Abstract

The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (ϳ5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (ϳ200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the ؊1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a ؊1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide.Recent research on in vitro primer extension reactions catalyzed by a range of DNA polymerases and using the hydrophobic T isostere 2,4-difluorotoluene (DFT) 3 (Fig. 1) and other analogs with substituents of increasing size at the 2-and 4-positions of the aromatic moiety appear to support different mechanisms of nucleotide insertion by high fidelity (A-class) and trans-lesion (Y-class) DNA polymerases (reviewed in Refs. 1 and 2). Thus, accurate replication by A-class polymerases may be more dependent on a close steric match between the active site and the shape of a Watson-Crick base pair ("active site tightness") than on the formation of complementary hydrogen bonds between incoming nucleotide base and template residue (3-6). Conversely, their more open active sites may render Y-class polymerases (6 -10) less sensitive to changes in base pair dimensions but more dependent on formation of hydrogen bonds between nucleotide pairs at the replicative position (6 -9). A-class DNA polymerases whose activities were assessed using apolar T analogs to date include Escherichia coli pol I (3-5) and the polymerase from phage T7 (6), and the tested Y-class DNA polymerases consisting of yeast pol (7), human pol (8), and the Dbh (DinB homolog (5)) and Dpo4 (9) polymerases from Sulfolobus acidocaldarius and Sulfolobus solfataricus, respectively.Careful review of the accu...

Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation

“…Additionally, the fact that there has not been a reported type II structure containing a 0 templating natural pyrimidine can arguably be considered evidence, by default, that the Ϫ1 frameshift mutation is unfavored by Dpo4 in these cases. Stacking potential and not necessarily a larger relative stacking surface may also lead to increased Ϫ1 frameshift mutations, as was inferred by the recently solved type II crystal structure of a 0 templating hydrophobic thymidine isostere 2Ј-deoxy-2,4-difluorotoluyl with an incoming dA:dGTP pair at the ϩ1 position (34). Overall, the data suggest that the energy supplied by the increased stacking surface and/or stacking potential of the 0 template base and the accompanying hydrogen bonds formed between the incoming base and ϩ1 base combine to provide sufficient stability to counter the large contortions of the active site that occur when the dNTP is positioned opposite the ϩ1 base.…”

Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase

“…A ternary complex was subsequently formed with the addition of 1 mM D -dCTP or its L -analogs: L -dCTP, (–)3TC-TP, (–)3TC-PPNP, (–)FTC-TP and (–)FTC-PPNP. Notably, non-catalytic metal ions Ca(II), rather than catalytic metal ions Mg(II), have been used regularly to trap incoming nucleotides in pre-insertion ternary complexes with Dpo4 (28–31) and other DNA polymerases (32–35). (–)3TC-TP and (–)FTC-TP were synthesized and purified by Gilead Sciences with >95% purity (HPLC).…”

Measurement and Theory of Hydrogen Bonding Contribution to Isosteric DNA Base Pairs

“…14 Two of the studies reported that the analog was not within hydrogen-bonding distance of adenine despite the opportunity to do so: Xia et al described an RNA duplex (measured at 1.61 Å resolution) containing the difluorotoluene base paired opposite adenine. 13a Although directly adjacent in a pairing configuration, the two “bases” were found not to approach one another within van der Waals distance, showing no evidence for attractive pairing interaction. Similarly, Irimia et al reported a co-crystal structure of the Dpo4 polymerase bound to DNA containing F opposite adenine (2.8 Å resolution).…”