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.

Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase

“…The contacts of Val 383 and Ile 384 with the nucleosides in the minor groove at the nick were proposed to be a principal factor in splaying apart the bases (8). The present mutational data suggest that this OB-induced distortion is a necessary component of the ligation pathway and are in accord with findings that Thermus LigA is sensitive to perturbations of the nick 3Ј-OH base pair in the minor groove (24).…”

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

“…6b) and actively maintains the B-form that is essential for efficient ligation. Consistent with this model, it has been shown that the presence of an A-form structure downstream of a nick severely affects ligation efficiency [17,23,24].…”

Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

“…The ability of ligases to ligate ends containing a mismatch, albeit with reduced efficiency, has been previously reported (48). To ensure we were measuring a "repair" reaction, we included [␣- 32 P]dCTP to monitor reactions that included the incorporation of dCMP to repair the T:G mismatch.…”