A human PMS2 homologue from Aquifex aeolicus stimulates an ATP-dependent DNA helicase.
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.
New England Biolabs Inc., Ipswich, Massachusetts 01938, USA.
Jerome
J
Mauris
Thomas C
TC
Evans
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001419:Bacteria / Q000378:metabolism*; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry* / Q000502:physiology; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000502:physiology; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D000070956:MutL Proteins; D009841:Oligonucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D009841:Oligonucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; C506746:UvrD protein, E coli; D000070976:Mismatch Repair Endonuclease PMS2; D000070956:MutL Proteins; D004265:DNA Helicases; D045643:DNA Repair Enzymes