Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

Abstract

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase (Pol) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Pol are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Pol; in this regard, Pol differs strikingly from classical high-fidelity DNA polymerases.Classical DNA polymerases, such as T7 and Escherichia coli Klenow, synthesize DNA with high fidelity, and they are unable to replicate through DNA lesions (7). By contrast, the eukaryotic polymerase (Pol) is a low-fidelity enzyme (16,28,45) with the ability to replicate through DNA lesions. Pol is unique among eukaryotic DNA polymerases in its proficient ability to replicate through UV-induced cyclobutane pyrimidine dimers (CPDs) (15). Remarkably, both yeast and human Pol insert A's opposite the two T's of a cis-syn thyminethymine (TT) dimer with the same efficiency and accuracy as they insert A's opposite undamaged template bases (16,44). In addition to TT dimers, UV light also induces the formation of CPDs at 5Ј-TC-3Ј and 5Ј-CC-3Ј sites, and genetic studies with Saccharomyces cerevisiae have indicated a role of Pol in the accurate bypass of these lesions as well (48). Because of its role in promoting the error-free bypass of CPDs, inactivation of Pol in humans causes UV hypermutability (43) and results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum (14,27). Yeast Pol also replicates through an 8-oxoguanine (8-oxoG) lesion efficiently and accurately, and genetic studies with S. cerevisiae have corroborated the requirement of Pol in the error-free bypass of this DNA lesion (11).From studies done with nonpolar isosteric analogs of natural DNA bases which lack the ability to form Watson-Crick (W-C) hydrogen (H) bonds, it has been concluded that W-C H bonding is not needed for DNA synthesis by classical high-fidelity DNA polymerases (30,32,33); rather, the geometric fit of the incoming nucleotide with the templating base within the polymerase active site is the principal determinant of the efficiency and fidelity of DNA synthesis in these polymerases (7,9,22). Here we examine the effects of difluorotoluene (F), which is virtually identical in shape, size, and conformation to thymine (T) but lacks the ability to form W-C H bonds with adenine (A) (Fig. 1A), on DNA synthesis by yeast Pol. Because of the inability of F to form W-C H bonds in water (37, 38), this compound is ideal for examining the relative importance of hydrogen bonds and steric effects on DNA synthesis. P...

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

“…Moreover, there is a significant asymmetry in the efficiency of the extension reaction, an ϳ30-fold reduction for insertion of dATP opposite template DFT and ϳ900-fold reduction for insertion of d(DFT)TP opposite template A (relative to the corresponding processes with T and dTTP, respectively (5)). The latter loss is comparable with the effects on insertion reactions involving the DFT analog seen with Y-class polymerases (5,(7)(8)(9). Together, these observations appear inconsistent with the conclusion that A-and Y-class polymerases use different mechanisms of replication and that the former may rely chiefly on shape for efficient and correct nucleotide insertion (1,2).…”

“…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)(4)(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.…”

Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase κ

“…Synthetic oliogodeoxynucleotide template and primers were used to prepare the nondamaged DNA substrates. The 3-deazaguanine (3DG)-and F-containing DNA substrates were synthesized and purified as described previously (37,45). For the 3DG-containing substrates, the following template/primer pairs were used.…”

“…We have shown previously that Pol differs from highfidelity replication/repair polymerases in its inability to replicate non-hydrogen-bonding nucleotide analogs. We hypothesized that this enzyme, lacking a tight geometric constraint, may rely instead upon Watson-Crick hydrogen bonding for the efficiency and fidelity of DNA synthesis (37).…”

Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

“…These results can be partially explained by base-pairing modes and the structures in the active site of each polymerase. For pols and , which require Watson-Crick base pairing for efficient catalysis (50,51), dCTP is more efficiently incorporated opposite N 2 -G adducts than O 6 -G adducts, because optimal Watson-Crick hydrogen bonding is possible only with N 2 -G adducts but not with O 6 -G adducts. This view would be consistent with recent reports that the S. solfataricus Y family DNA polymerase Dpo4 forms a wobble base pair between C and O 6 -MeG (or O 6 -BzG), and thus the polymerization is inhibited (52,53 may uniquely utilize an Arg for the optimal hydrogen bonding with an incoming dCTP, as shown with yeast Rev1 (34).…”