A human PMS2 homologue from Aquifex aeolicus stimulates an ATP-dependent DNA helicase

Abstract

Mismatch repair in Escherichia coli involves a number of proteins including MutL and UvrD. Eukaryotes also possess MutL homologues; however, no UvrD helicase homologues have been identified. The hyperthermophilic bacterium Aquifex aeolicus has a MutL protein (Aae MutL) that possesses a latent endonuclease activity similar to eukaryotic, but different from E. coli, MutL proteins. By sequence homology Aq793 is a member of the PcrA/UvrD/Rep helicase subfamily. We expressed Aae MutL and the putative A. aeolicus DNA helicase (Aq793) proteins in E. coli. Using synthetic oligonucleotide substrates, we observed that lower concentrations of Aq793 were required to unwind double-stranded DNA that had a 3'-poly(dT) overhang as compared with double-stranded DNA with a 5'-poly(dT) or lacking a poly(dT) tail. This unwinding activity was stimulated by adding Aae MutL with maximal stimulation observed at an approximately 1.5:1 (MutL:Aq793) stoichiometric ratio. The enhancement of Aq793 helicase activity did not require the Aae MutL protein to retain endonuclease activity. Furthermore, the C-terminal 123 amino acid residues of Aae MutL were sufficient to stimulate Aq793 helicase activity, albeit at a significantly reduced efficacy. To the best of our knowledge this is the first time a human PMS2 homologue has been demonstrated to stimulate a PcrA/UvrD/Rep subfamily helicase, and this finding may further our understanding of the evolution of the mismatch repair pathway.

FIGURE 1.

Aq793 helicase activity. DNA unwinding activity was assayed with serial dilutions of Aq793. The 20-μl unwinding reactions were incubated at 55 °C and contained 0.02 nm dsDNA substrate. The reactions were quenched after 20 min using stop solution and applied to a 20% TBE acrylamide gel as described under “Materials and Methods.” A, representative autoradiograms of TBE acrylamide gels. The DNA substrate is indicated to the left of the gel picture, and the expected mobility of dsDNA and ssDNA is shown to the right. The negative control (−) was performed using 0.02 nm of substrate incubated at 55 °C with 400 nm of Aq793 without ATP, and the positive control (+) was performed at 95 °C in the absence of Aq793. B, graph of data from TBE gel bands that were quantified using ImageQuant TL software. 3′-dT40 (solid squares), 5′-dT40 (open circles), dT40⁻ (open triangles), and the nicked substrate (solid diamonds) are represented. The points represent the averages of three separate experiments. The error bars represent the standard deviation at each point.

FIGURE 2.

Aae MutL stimulation of Aq793 helicase activity. Serial dilutions of Aae MutL were incubated with a fixed concentration of Aq793 (23.5 pm) as described under “Materials and Methods.” The reactions were performed for 20 min at 55 °C and contained 0.02 nm duplex DNA substrate. The samples were resolved on a 20% TBE acrylamide gel and visualized by autoradiography. A, a representative TBE acrylamide gel of the reaction containing 3′-dT40 duplex DNA substrate. The expected mobility of dsDNA and ssDNA are indicated to the right of the picture. The negative control (−) contained Aq793 helicase but lacked MutL. B, graphical representation of the TBE acrylamide gel data. The gel bands were quantified using ImageQuant TL software, and the individual points represent the averages of three separate experiments, and the error bars show the standard deviation at each point. 3′-dT40 (solid squares), 5′-dT40 (open circles), and dT40⁻ (open triangles) DNA substrates are shown.

FIGURE 3.

Aae MutL (E357K) and Aae MutL-CTD stimulate Aq793 helicase activity. Aq793 was assayed for unwinding activity in the presence of serial dilutions of Aae MutL (E357K) and Aae MutL-CTD. The reactions were performed as described in the legend to Fig. 2 and under “Materials and Methods.” A, representative TBE acrylamide gel of the titration of Aae MutL-CTD (top gel picture) and Aae MutL (E357K) (bottom gel picture) mutants. Negative controls (−) contained Aq793 protein but not the respective MutL mutant. B, graphical representation of the effect of Aae MutL-CTD (solid squares) and Aae MutL (E357K) (open circles) on the oligonucleotide displacement activity of Aq793. Gel bands were quantified using ImageQuant TL software, and individual points represent the averages of three separate experiments.

FIGURE 4.

Aae MutL stimulation of Aq793 helicase activity on a nicked substrate. Serial dilutions of Aae MutL were incubated with a fixed concentration of Aq793 (1 nm) as described under “Materials and Methods.” The reactions were performed for 20 min at 55 °C and contained 0.02 nm duplex DNA substrate. The samples were resolved on a 20% TBE acrylamide gel and visualized by autoradiography. A, a representative TBE acrylamide gel of the reaction containing nicked DNA substrate. The expected mobility of dsDNA and ssDNA are indicated to the right of the picture. The negative control (−) contained Aq793 helicase but lacked MutL. B, a graphical representation of the TBE acrylamide gel data. The gel bands were quantified using ImageQuant TL software. The individual points represent the averages of three separate experiments, and the error bars show the standard deviation at each point.