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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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Hydrolysis efficiency of oligonucleotide substrates is affected by the location of the guanine tract.The relative hydrolysis efficacy of each oligomer is indicated by the percentage recovery of their constituent 5′dNMPs. (A) 300 µM oligomer substrates containing either no guanine (G-NO) or guanine tracts at the ends (G2-CAP ) or in the middle (G4-MID) of the polynucleotide chain were incubated at 37°C for twenty-four hours with 0.5 U BAL 31 enzyme. (B) The same experiment as in (A) except using 30 µM oligomers and an incubation temperature of 55°C.
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pone-0003595-g003: Hydrolysis efficiency of oligonucleotide substrates is affected by the location of the guanine tract.The relative hydrolysis efficacy of each oligomer is indicated by the percentage recovery of their constituent 5′dNMPs. (A) 300 µM oligomer substrates containing either no guanine (G-NO) or guanine tracts at the ends (G2-CAP ) or in the middle (G4-MID) of the polynucleotide chain were incubated at 37°C for twenty-four hours with 0.5 U BAL 31 enzyme. (B) The same experiment as in (A) except using 30 µM oligomers and an incubation temperature of 55°C.

Mentions: The oligomers G-NO, G2-CAP, and G4-MID were incubated at 37°C with 0.5 U enzyme for a twenty-four hour period (in quintuplet) and, as before, their hydrolysis efficiency was measured by the recovery of the constituent nucleotides. An ANOVA analysis was conducted to determine whether the variation in recovery rates were significant. The G2-CAP oligomer produced nucleotides at recovery rates similar to those seen with the non-G containing homopolymeric oligomers and with the G-NO oligomer (Fig. 3A). In contrast, G4-MID produced non-G nucleotides at significantly lower recovery rates compared to G2-CAP. G-tract hydrolysis, as measured by the formation of dGMP, occurred with both G2-CAP and G4-MID, but at lower amounts than the other three nucleotides. Thus G di-nucleotides at the 5′ and 3′ ends of decameric oligonucleotides appear to be permissive for BAL 31 digestion of the decamer, whereas a G tetra-nucleotide tract in the middle of the decamer makes it more refractory to hydrolysis. This possibly indicates that the hydrolysis proceeds from one end of the ssDNA and/or that guanine tracts are difficult to fully hydrolyze.


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

Marrone A, Ballantyne J - PLoS ONE (2008)

Hydrolysis efficiency of oligonucleotide substrates is affected by the location of the guanine tract.The relative hydrolysis efficacy of each oligomer is indicated by the percentage recovery of their constituent 5′dNMPs. (A) 300 µM oligomer substrates containing either no guanine (G-NO) or guanine tracts at the ends (G2-CAP ) or in the middle (G4-MID) of the polynucleotide chain were incubated at 37°C for twenty-four hours with 0.5 U BAL 31 enzyme. (B) The same experiment as in (A) except using 30 µM oligomers and an incubation temperature of 55°C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570716&req=5

pone-0003595-g003: Hydrolysis efficiency of oligonucleotide substrates is affected by the location of the guanine tract.The relative hydrolysis efficacy of each oligomer is indicated by the percentage recovery of their constituent 5′dNMPs. (A) 300 µM oligomer substrates containing either no guanine (G-NO) or guanine tracts at the ends (G2-CAP ) or in the middle (G4-MID) of the polynucleotide chain were incubated at 37°C for twenty-four hours with 0.5 U BAL 31 enzyme. (B) The same experiment as in (A) except using 30 µM oligomers and an incubation temperature of 55°C.
Mentions: The oligomers G-NO, G2-CAP, and G4-MID were incubated at 37°C with 0.5 U enzyme for a twenty-four hour period (in quintuplet) and, as before, their hydrolysis efficiency was measured by the recovery of the constituent nucleotides. An ANOVA analysis was conducted to determine whether the variation in recovery rates were significant. The G2-CAP oligomer produced nucleotides at recovery rates similar to those seen with the non-G containing homopolymeric oligomers and with the G-NO oligomer (Fig. 3A). In contrast, G4-MID produced non-G nucleotides at significantly lower recovery rates compared to G2-CAP. G-tract hydrolysis, as measured by the formation of dGMP, occurred with both G2-CAP and G4-MID, but at lower amounts than the other three nucleotides. Thus G di-nucleotides at the 5′ and 3′ ends of decameric oligonucleotides appear to be permissive for BAL 31 digestion of the decamer, whereas a G tetra-nucleotide tract in the middle of the decamer makes it more refractory to hydrolysis. This possibly indicates that the hydrolysis proceeds from one end of the ssDNA and/or that guanine tracts are difficult to fully hydrolyze.

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