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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 titration of Aae MutL.A, Representative data for the nicking of supercoiled pBR322 by various Aae MutL concentrations. The reactions were performed in the presence of 1 mM Mn++ and with (left hand panel) or without (right hand panel) 1 mM ATP. The reaction products were resolved on a 1% agarose gel and supercoiled (SC), nicked (N), and linear (L) forms of the plasmid were visible. The control reaction (-) contained supercoiled pBR322 incubated for 1 hour at 37°C in the absence of MutL. The reactions with 2-fold serial dilutions of MutL from 2600 nM (lane 2) to 0.159 nM (lane 16) were incubated for 1 hour at 37°C. B, Graphical representation of agarose gel data showing the relaxation of pBR322 by different amounts of Aae MutL. The enzyme titration was performed as in Figure 1A with 1 mM Mn++ (solid triangle), 1 mM ATP (solid square), or 1 mM Mn++ and 1 mM ATP (open square). Each point represents the average of 3 separate experiments.
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pone-0007175-g001: Endonuclease activity titration of Aae MutL.A, Representative data for the nicking of supercoiled pBR322 by various Aae MutL concentrations. The reactions were performed in the presence of 1 mM Mn++ and with (left hand panel) or without (right hand panel) 1 mM ATP. The reaction products were resolved on a 1% agarose gel and supercoiled (SC), nicked (N), and linear (L) forms of the plasmid were visible. The control reaction (-) contained supercoiled pBR322 incubated for 1 hour at 37°C in the absence of MutL. The reactions with 2-fold serial dilutions of MutL from 2600 nM (lane 2) to 0.159 nM (lane 16) were incubated for 1 hour at 37°C. B, Graphical representation of agarose gel data showing the relaxation of pBR322 by different amounts of Aae MutL. The enzyme titration was performed as in Figure 1A with 1 mM Mn++ (solid triangle), 1 mM ATP (solid square), or 1 mM Mn++ and 1 mM ATP (open square). Each point represents the average of 3 separate experiments.

Mentions: As expected, Aae MutL nicking activity required the presence of Mn++ (Figure 1). The nicking assay was performed in the presence or absence of ATP at constant pBR322 concentration for 1 hour at 37°C with Aae MutL serially diluted from 2.6 µM to 0.16 nM (Figure 1). Reaction outcomes were visualized by application to an agarose gel containing ethidium bromide (Figure 1A). Agarose gel band intensities were quantified using ImageJ® software and the data represented graphically (Figure 1B). As expected the conversion of the supercoiled pBR322 to the nicked form occurred at lower Aae MutL concentrations in the absence of ATP as compared to the presence of ATP (Figure 1B). However, at the higher Aae MutL concentrations in the presence of ATP the plasmid was highly degraded when compared to equivalent enzyme concentrations in the absence of ATP. Typically, increased plasmid degradation indicates increased non-specific endonuclease activity.


Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

Mauris J, Evans TC - PLoS ONE (2009)

Endonuclease activity titration of Aae MutL.A, Representative data for the nicking of supercoiled pBR322 by various Aae MutL concentrations. The reactions were performed in the presence of 1 mM Mn++ and with (left hand panel) or without (right hand panel) 1 mM ATP. The reaction products were resolved on a 1% agarose gel and supercoiled (SC), nicked (N), and linear (L) forms of the plasmid were visible. The control reaction (-) contained supercoiled pBR322 incubated for 1 hour at 37°C in the absence of MutL. The reactions with 2-fold serial dilutions of MutL from 2600 nM (lane 2) to 0.159 nM (lane 16) were incubated for 1 hour at 37°C. B, Graphical representation of agarose gel data showing the relaxation of pBR322 by different amounts of Aae MutL. The enzyme titration was performed as in Figure 1A with 1 mM Mn++ (solid triangle), 1 mM ATP (solid square), or 1 mM Mn++ and 1 mM ATP (open square). Each point represents the average of 3 separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007175-g001: Endonuclease activity titration of Aae MutL.A, Representative data for the nicking of supercoiled pBR322 by various Aae MutL concentrations. The reactions were performed in the presence of 1 mM Mn++ and with (left hand panel) or without (right hand panel) 1 mM ATP. The reaction products were resolved on a 1% agarose gel and supercoiled (SC), nicked (N), and linear (L) forms of the plasmid were visible. The control reaction (-) contained supercoiled pBR322 incubated for 1 hour at 37°C in the absence of MutL. The reactions with 2-fold serial dilutions of MutL from 2600 nM (lane 2) to 0.159 nM (lane 16) were incubated for 1 hour at 37°C. B, Graphical representation of agarose gel data showing the relaxation of pBR322 by different amounts of Aae MutL. The enzyme titration was performed as in Figure 1A with 1 mM Mn++ (solid triangle), 1 mM ATP (solid square), or 1 mM Mn++ and 1 mM ATP (open square). Each point represents the average of 3 separate experiments.
Mentions: As expected, Aae MutL nicking activity required the presence of Mn++ (Figure 1). The nicking assay was performed in the presence or absence of ATP at constant pBR322 concentration for 1 hour at 37°C with Aae MutL serially diluted from 2.6 µM to 0.16 nM (Figure 1). Reaction outcomes were visualized by application to an agarose gel containing ethidium bromide (Figure 1A). Agarose gel band intensities were quantified using ImageJ® software and the data represented graphically (Figure 1B). As expected the conversion of the supercoiled pBR322 to the nicked form occurred at lower Aae MutL concentrations in the absence of ATP as compared to the presence of ATP (Figure 1B). However, at the higher Aae MutL concentrations in the presence of ATP the plasmid was highly degraded when compared to equivalent enzyme concentrations in the absence of ATP. Typically, increased plasmid degradation indicates increased non-specific endonuclease activity.

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