title: Inefficient excision of uracil from loop regions of DNA oligomers by E. coli uracil DNA glycosylase.
abstract: Kinetic parameters for uracil DNA glycosylase (E. coli)-catalysed excision of uracil from DNA oligomers containing dUMP in different structural contexts were determined. Our results show that single-stranded oligonucleotides (unstructured) are used as somewhat better substrates than the double-stranded oligonucleotides. This is mainly because of the favourable Vmax value of the enzyme for single-stranded substrates. More interestingly, however, we found that uracil release from loop regions of DNA hairpins is extremely inefficient. The poor efficiency with which uracil is excised from loop regions is a result of both increased Km and lowered Vmax values. This observation may have significant implications in uracil DNA glycosylase-directed repair of DNA segments that can be extruded as hairpins. In addition, these studies are useful in designing oligonucleotides for various applications in DNA research where the use of uracil DNA glycosylase is sought.
authors.affiliation: Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
authors.forename: N V
authors.initials: NV
authors.lastname: Kumar
authors.forename: U
authors.initials: U
authors.lastname: Varshney
mesh_terms: D001483:Base Sequence; D045647:DNA Glycosylases; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
chemical_list: D004277:DNA, Single-Stranded; D011089:Polydeoxyribonucleotides; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase