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


RESULTS
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.
Marrone
April
A
Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, Orlando, Florida, USA.
Ballantyne
Jack
J
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004526:Efficiency; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005813:Genes, Synthetic; D006147:Guanine / Q000378:metabolism*; D006868:Hydrolysis; D046911:Macromolecular Substances / Q000378:metabolism; D011108:Polymers / Q000138:chemical synthesis / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D011108:Polymers; D006147:Guanine; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31