sequence specificity of bal 31 nuclease for ssdna revealed by synthetic oligomer substrates containing homopolymeric guanine tracts序列特异性bal 31 ssdna透露的核酸酶合成的低聚物基质含有homopolymeric鸟嘌呤大片.pdfVIP

Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts April Marrone1,2, Jack Ballantyne1,2* 1 Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, Orlando, Florida, United States of America, 2 National Center for Forensic Science, Orlando, Florida, United States of America Abstract Background: The extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 59-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 39 end of ten and twenty base oligomers. GNC 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 GNC 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 sp