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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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Aae MutL adenine nucleotide binding.The affinity of Aae MutL for different adenine nucleotides was measured by a filter binding assay. The binding of Aae MutL to ATP (open circles) or ADP (solid squares) or of Aae MutL LC20 to ATP (open triangles) was measured. A, nucleotide binding in the presence of dsDNA. B, Influence of ssDNA on full-length Aae MutL nucleotide binding. C, the binding of Aae MutL and Aae MutL LC20 to adenine nucleotides in the absence of added DNA. Each point represents the average of 3 separate experiments.
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pone-0007175-g006: Aae MutL adenine nucleotide binding.The affinity of Aae MutL for different adenine nucleotides was measured by a filter binding assay. The binding of Aae MutL to ATP (open circles) or ADP (solid squares) or of Aae MutL LC20 to ATP (open triangles) was measured. A, nucleotide binding in the presence of dsDNA. B, Influence of ssDNA on full-length Aae MutL nucleotide binding. C, the binding of Aae MutL and Aae MutL LC20 to adenine nucleotides in the absence of added DNA. Each point represents the average of 3 separate experiments.

Mentions: A filter-binding assay was used to investigate Aae MutL nucleotide binding alone or in the presence of ssDNA or dsDNA (Figure 6). In the absence of DNA or in the presence of ssDNA MutL binds both ATP and ADP with about equal affinity. In the presence of phage M13 dsDNA, however MutL displayed an approximately 2-fold increase in affinity for ATP.


Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

Mauris J, Evans TC - PLoS ONE (2009)

Aae MutL adenine nucleotide binding.The affinity of Aae MutL for different adenine nucleotides was measured by a filter binding assay. The binding of Aae MutL to ATP (open circles) or ADP (solid squares) or of Aae MutL LC20 to ATP (open triangles) was measured. A, nucleotide binding in the presence of dsDNA. B, Influence of ssDNA on full-length Aae MutL nucleotide binding. C, the binding of Aae MutL and Aae MutL LC20 to adenine nucleotides in the absence of added DNA. 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-g006: Aae MutL adenine nucleotide binding.The affinity of Aae MutL for different adenine nucleotides was measured by a filter binding assay. The binding of Aae MutL to ATP (open circles) or ADP (solid squares) or of Aae MutL LC20 to ATP (open triangles) was measured. A, nucleotide binding in the presence of dsDNA. B, Influence of ssDNA on full-length Aae MutL nucleotide binding. C, the binding of Aae MutL and Aae MutL LC20 to adenine nucleotides in the absence of added DNA. Each point represents the average of 3 separate experiments.
Mentions: A filter-binding assay was used to investigate Aae MutL nucleotide binding alone or in the presence of ssDNA or dsDNA (Figure 6). In the absence of DNA or in the presence of ssDNA MutL binds both ATP and ADP with about equal affinity. In the presence of phage M13 dsDNA, however MutL displayed an approximately 2-fold increase in affinity for ATP.

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