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Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

Mauris J, Evans TC - PLoS ONE (2009)

Bottom Line: Mn(++) was previously found to stimulate the endonuclease activity of these homologues.A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA.Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc., Ipswich, Massachusetts, USA.

ABSTRACT

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.

Methodologies/principal findings: 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.

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Endonuclease activity of the Aae MutL LC20 fragment.Different concentrations of the Aae MutL C-terminal domain were incubated with 3.5 nM of pBR322 plasmid. The reactions were incubated for an hour at 37°C and stopped by adding loading buffer as described in the Materials and Methods. Reaction products were resolved on a 1% agarose gel. The gel picture was analyzed with ImageJ software. The percentage of nicked plasmid was plotted against the log of the protein concentration. The reaction was performed with 1 mM Mn++ (solid triangle), with 1 mM ATP (solid square), or with 1 mM ATP and 1 mM Mn++(open square)
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pone-0007175-g005: Endonuclease activity of the Aae MutL LC20 fragment.Different concentrations of the Aae MutL C-terminal domain were incubated with 3.5 nM of pBR322 plasmid. The reactions were incubated for an hour at 37°C and stopped by adding loading buffer as described in the Materials and Methods. Reaction products were resolved on a 1% agarose gel. The gel picture was analyzed with ImageJ software. The percentage of nicked plasmid was plotted against the log of the protein concentration. The reaction was performed with 1 mM Mn++ (solid triangle), with 1 mM ATP (solid square), or with 1 mM ATP and 1 mM Mn++(open square)

Mentions: Incubation of the Aae MutL-CTD in the absence of Mn++ resulted in no detectable relaxation of pBR322 (Figure 5). Addition of 1 mM Mn++ to the reaction, however, induced a latent endonuclease activity that was able to completely relax the supercoiled pBR322 at the higher enzyme concentrations. Not surprisingly, the specific activity of the Aae MutL-CTD was apparently lower than the full-length protein because approximately 700 times more Aae MutL-CTD was needed to see plasmid relaxation as compared to the full-length Aae MutL. Unlike the full-length enzyme, Aae MutL-CTD did not display detectable differences in behavior in the presence or absence of ATP (Figure 5). The Mn++ induced endonuclease activity of Aae MutL-CTD was observed when the reactions were performed at either 37°C (Figure 5) or 55°C (Figure S2).


Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

Mauris J, Evans TC - PLoS ONE (2009)

Endonuclease activity of the Aae MutL LC20 fragment.Different concentrations of the Aae MutL C-terminal domain were incubated with 3.5 nM of pBR322 plasmid. The reactions were incubated for an hour at 37°C and stopped by adding loading buffer as described in the Materials and Methods. Reaction products were resolved on a 1% agarose gel. The gel picture was analyzed with ImageJ software. The percentage of nicked plasmid was plotted against the log of the protein concentration. The reaction was performed with 1 mM Mn++ (solid triangle), with 1 mM ATP (solid square), or with 1 mM ATP and 1 mM Mn++(open square)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2744016&req=5

pone-0007175-g005: Endonuclease activity of the Aae MutL LC20 fragment.Different concentrations of the Aae MutL C-terminal domain were incubated with 3.5 nM of pBR322 plasmid. The reactions were incubated for an hour at 37°C and stopped by adding loading buffer as described in the Materials and Methods. Reaction products were resolved on a 1% agarose gel. The gel picture was analyzed with ImageJ software. The percentage of nicked plasmid was plotted against the log of the protein concentration. The reaction was performed with 1 mM Mn++ (solid triangle), with 1 mM ATP (solid square), or with 1 mM ATP and 1 mM Mn++(open square)
Mentions: Incubation of the Aae MutL-CTD in the absence of Mn++ resulted in no detectable relaxation of pBR322 (Figure 5). Addition of 1 mM Mn++ to the reaction, however, induced a latent endonuclease activity that was able to completely relax the supercoiled pBR322 at the higher enzyme concentrations. Not surprisingly, the specific activity of the Aae MutL-CTD was apparently lower than the full-length protein because approximately 700 times more Aae MutL-CTD was needed to see plasmid relaxation as compared to the full-length Aae MutL. Unlike the full-length enzyme, Aae MutL-CTD did not display detectable differences in behavior in the presence or absence of ATP (Figure 5). The Mn++ induced endonuclease activity of Aae MutL-CTD was observed when the reactions were performed at either 37°C (Figure 5) or 55°C (Figure S2).

Bottom Line: Mn(++) was previously found to stimulate the endonuclease activity of these homologues.A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA.Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc., Ipswich, Massachusetts, USA.

ABSTRACT

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.

Methodologies/principal findings: 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.

Show MeSH
Related in: MedlinePlus