DNA ligases ensure fidelity by interrogating minor groove contacts

Abstract

DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3' side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson-Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson-Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson-Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3' side of the ligase junction. The NAD+-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.

Figure 1

Proposed conformations of the analog base pairs employed in this study. The abbreviations of the analogs are as follows: U, uracil; G, guanine; Hx, hypoxanthine (deoxyinosine); R, nebularine (purine); A, adenine; 2AP, 2-aminopurine; DAP, 2,6-diaminopurine. U:A, U:2AP and U:DAP are of normal Watson–Crick geometry. U:R is stabilized by one hydrogen-bonding interaction in a pseudo Watson–Crick geometry. U:G and U:Hx exhibit wobble geometry [adapted from (28)].

Figure 2

Tth ligase activity on terminal U:purine base pairs on the 3′-hydroxyl end (A) and the 5′-phosphate end (B) of a ligase junction. The gel pictures were performed at 26.5°C for 30 min with 0.5 pmol of duplexes and 10 U of Tth DNA ligase in a total volume of 10 μl. AppDNA indicates the 5′-AMP intermediate of the ligation reaction. The sequences used are listed above the gel pictures, where X = A, DAP, 2AP, R, G and Hx. In order to keep the surrounding sequence constant, the ligase junction was shifted by one base when U was moved from the 3′-hydroxyl end to the 5′-phosphate end. The gray bars represent the ligation rates (femtomoles ligated per minute per unit ligase), which were averaged from two independent measurements. 3′ (5′)-X:Y indicates that X is on the 3′-hydroxyl (5′-phosphate) end of the ligase junction, and Y is on the template.

Figure 3

pH-dependent equilibrium between neutral wobble and ionized Watson-Crick structures of FU:G base pair. The pK_a value of the N3 proton of FU is 8.3 (32).

Figure 4

Sample experiments of the ligation time course for (A) 3′-C:G, (B) 3′-FU:G and (C) 3′-U:G as a function of pH. Time courses at pH 7.1 (closed diamond) and pH 9 (open square) are shown. Fraction ligated was averaged from three independent measurements.

Figure 5

The ligation rates of Tth ligase on FU:G (open triangle) and U:G (shaded square) as a function of solution pH. Results on the 3′ end of the ligase junction are presented in (A) while on the 5′-end in (B). Ligation efficiency is the ligation rate normalized to that of C:G, which is shown in the insets. Sequences used are listed where X = U or FU.

Figure 6

DNA ligase assays with substrates containing DFT. (A) The structure of DFT is shown above the gel, and hydrogen bonding acceptors in the minor groove of an A:T pair are indicated by arrows. (B) Tth DNA ligase assays with DFT on the 5′-phosphate end of a ligase junction. The experiments were conducted at 26.5°C for 30 min with 0.5 pmol of duplexes and 10 U of Tth DNA ligase in a total volume of 10 μl. Control lanes contain no protein. (C) Tth and T4 DNA ligase assays with DFT on the 3′-hydroxyl end of a ligase junction. Both ligase experiments were conducted with equimolar DNA duplex and enzyme (100 fmol each) in a total volume of 20 μl. Tth ligase reactions were incubated at 26.5°C for 5 min, and T4 ligase at 16°C for 10 min in the corresponding reaction buffer as described in Materials and Methods. 3′ (5′)-X:Y indicates that X is on the 3′-hydroxyl (5′-phosphate) end of the ligase junction, and Y is on the template.

Figure 7

EMSA of Tth DNA ligase bound to duplex DNA. (A) Effects of minor groove interactions as well as Mg²⁺ on Tth ligase binding and ligation. DNA samples (100 fmol) were incubated with 100 fmol of Tth DNA ligase at 26.5°C in the presence or absence of Mg²⁺ in 20 μl total volume. After 5 min incubation, half of the samples were adjusted to 10% glycerol, and electrophoresed through a 10% native gel to detect ligase binding. The other half were heat-denatured, and resolved on a 20% denaturing gel to detect ligation. (B) Tth DNA ligase binds to duplexes containing the uracil:purine base pair on the 3′-hydroxyl end. The experiments were performed with equimolar DNA substrates and the ligase (100 fmol each) at 26.5°C for 5 min in the absence of Mg²⁺. 3′ (5′)-X:Y indicates that X is on the 3′-hydroxyl (5′-phosphate) end of the ligase junction, and Y is on the template.