A Human PMS2 Homologue from Aquifex aeolicus Stimulates an ATP-dependent DNA Helicase

• Published in: The Journal of biological chemistry 2010-04, Vol.285 (15), p.11087-11092
• Main Authors: Mauris, Jerome, Evans, Thomas C.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - physiology; Adenosine Triphosphate - chemistry; Amino acids; Aquifex aeolicus; Bacteria - metabolism; Base Pair Mismatch; DNA - chemistry; DNA and Chromosomes; DNA helicase; DNA Helicases - chemistry; DNA Helicases - physiology; DNA Repair; DNA Repair Enzymes - chemistry; DNA-Binding Proteins - chemistry; DNA/Damage; DNA/Enzymes; DNA/Protein Interaction; DNA/Uvr ABCD System; Endonuclease; Endonucleases - metabolism; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins - physiology; Evolution; Helicase; Homology; Humans; mismatch repair; Mismatch Repair Endonuclease PMS2; MutL; MutL protein; MutL Proteins; Oligonucleotides; Oligonucleotides - chemistry; Polyadenylation; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Unwinding
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.050955

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 ∼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.