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Sequence specificity of BAL 31 nuclease for ssDNA revealed by synthetic oligomer substrates containing homopolymeric guanine tracts.

Marrone A, Ballantyne J - PLoS ONE (2008)

Bottom Line: G*C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access.Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G*C rich regions.Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, Orlando, Florida, USA.

ABSTRACT

Background: The extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 5'-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites.

Principal findings: In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single-stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine's presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 3' end of ten and twenty base oligomers. G*C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G*C rich regions. Thus the presence of a guanine tract per se impairs the enzyme's functionality, possibly due to the tract's bulky nature and preventing efficient progression through the active site.

Conclusions: This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.

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G10 homodecamers continue to be refractory to hydrolysis at elevated temperatures.G10 and T10 were hydrolyzed by BAL 31 at 45°C, 50°C, and 55°C for a twenty-four hour period. The relative hydrolysis efficacy of each homopolymeric oligomer is indicated by the percentage recovery of their constituent 5′dNMPs.
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pone-0003595-g002: G10 homodecamers continue to be refractory to hydrolysis at elevated temperatures.G10 and T10 were hydrolyzed by BAL 31 at 45°C, 50°C, and 55°C for a twenty-four hour period. The relative hydrolysis efficacy of each homopolymeric oligomer is indicated by the percentage recovery of their constituent 5′dNMPs.

Mentions: We considered the possibility that the dramatic reduction in hydrolysis efficiency with dG10 could be an artifact due to guanine homoploymer self aggregation to form higher order structures [8], [9] that may not be efficient substrates for the enzyme. Lowering the salt concentration or increasing the temperature might be expected to decrease such aggregation. However, lowering the salt concentration would be counter productive due to the requirement of Mg2+ and Ca2+ for enzymatic activity. Since the enzyme requires a large deactivation temperature (∼85°C) and presumably is still active at elevated temperatures, the enzyme reaction temperature was increased from 37°C to 45°C, 50°C, and 55°C and the dG10 oligomers were incubated over a twenty-four hour period with 0.5 U enzyme. DT10 oligomers were incubated under the same conditions as a control. Samples were prepared in quintuplet. The dT10 samples produced 82.6±9.9%, 88.2±5.9%, and 98.4±8.4% dTMP at 45°C, 50°C, and 55°C respectively. The dG10 samples produced 0±0%, 1.31±0.94%, and 0.78±0.46% dGMP at 45°C, 50°C, and 55°C respectively (Fig. 2). Even if not all secondary structure was eliminated at these elevated temperatures, it would be alleviated which should lead to an increase in dGMP produced. These results indicated that the refractory nature of dG10 to BAL 31 digestion was probably not due to the self aggregation of homoguanosine oligomers into higher order structures.


Sequence specificity of BAL 31 nuclease for ssDNA revealed by synthetic oligomer substrates containing homopolymeric guanine tracts.

Marrone A, Ballantyne J - PLoS ONE (2008)

G10 homodecamers continue to be refractory to hydrolysis at elevated temperatures.G10 and T10 were hydrolyzed by BAL 31 at 45°C, 50°C, and 55°C for a twenty-four hour period. The relative hydrolysis efficacy of each homopolymeric oligomer is indicated by the percentage recovery of their constituent 5′dNMPs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570716&req=5

pone-0003595-g002: G10 homodecamers continue to be refractory to hydrolysis at elevated temperatures.G10 and T10 were hydrolyzed by BAL 31 at 45°C, 50°C, and 55°C for a twenty-four hour period. The relative hydrolysis efficacy of each homopolymeric oligomer is indicated by the percentage recovery of their constituent 5′dNMPs.
Mentions: We considered the possibility that the dramatic reduction in hydrolysis efficiency with dG10 could be an artifact due to guanine homoploymer self aggregation to form higher order structures [8], [9] that may not be efficient substrates for the enzyme. Lowering the salt concentration or increasing the temperature might be expected to decrease such aggregation. However, lowering the salt concentration would be counter productive due to the requirement of Mg2+ and Ca2+ for enzymatic activity. Since the enzyme requires a large deactivation temperature (∼85°C) and presumably is still active at elevated temperatures, the enzyme reaction temperature was increased from 37°C to 45°C, 50°C, and 55°C and the dG10 oligomers were incubated over a twenty-four hour period with 0.5 U enzyme. DT10 oligomers were incubated under the same conditions as a control. Samples were prepared in quintuplet. The dT10 samples produced 82.6±9.9%, 88.2±5.9%, and 98.4±8.4% dTMP at 45°C, 50°C, and 55°C respectively. The dG10 samples produced 0±0%, 1.31±0.94%, and 0.78±0.46% dGMP at 45°C, 50°C, and 55°C respectively (Fig. 2). Even if not all secondary structure was eliminated at these elevated temperatures, it would be alleviated which should lead to an increase in dGMP produced. These results indicated that the refractory nature of dG10 to BAL 31 digestion was probably not due to the self aggregation of homoguanosine oligomers into higher order structures.

Bottom Line: G*C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access.Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G*C rich regions.Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, Orlando, Florida, USA.

ABSTRACT

Background: The extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 5'-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites.

Principal findings: In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single-stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine's presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 3' end of ten and twenty base oligomers. G*C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G*C rich regions. Thus the presence of a guanine tract per se impairs the enzyme's functionality, possibly due to the tract's bulky nature and preventing efficient progression through the active site.

Conclusions: This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.

Show MeSH
Related in: MedlinePlus