Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.
BACKGROUND
Human PMS2 (hPMS2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++) was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)(2)E(X)(4)E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.


RESULTS
We examined the effect ATP had on the Mn(++) induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5) s(-1) and 4.2+/-0.3x10(-5) s(-1) in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)(2)E(X)(4)E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.


CONCLUSIONS
ATP stimulated the Mn(++) induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)(2)E(X)(4)E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++) induced nicking activity.
Mauris
Jerome
J
New England Biolabs, Inc., Ipswich, Massachusetts, USA.
Evans
Thomas C
TC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008345:Manganese / Q000737:chemistry; D000070976:Mismatch Repair Endonuclease PMS2; D008969:Molecular Sequence Data; D000070956:MutL Proteins; D009363:Neoplasm Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D009363:Neoplasm Proteins; C088996:PMS1 protein, human; D008345:Manganese; D000255:Adenosine Triphosphate; D004720:Endonucleases; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070956:MutL Proteins; D045643:DNA Repair Enzymes