A kinetic analysis of substrate recognition by uracil-DNA glycosylase from herpes simplex virus type 1.
Uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA. It has previously been demonstrated that UDG exhibits some sequence dependence in its activity, although this has not been well characterised. This study has investigated the sequence-dependent activity of UDG from herpes simplex virus type-1 (HSV-1). A more detailed analysis has been possible by using both kinetic and binding assays with a variety of different oligonucleotide substrates. The target uracil has been placed in substrates with either A-T-rich or G-C-rich flanking sequences and analyses have been performed on both the single- and double-stranded forms of each substrate. In the latter the uracil has been placed in both a U.A base pair and a U.G mismatch. It is observed that the sequences flanking the target uracil have a greater effect on UDG activity than the partner base of the uracil. Furthermore, the sequence context effects extend to single-stranded DNA. Systematic examination of the kinetics and binding of UDG with these different substrates has enabled us to examine the origin of the sequence preferences. We conclude that the damage recognition step in the HSV-1 UDG reaction pathway is modulated by local DNA sequence.
Bellamy
S R
SR
Imperial College of Science, Technology and Medicine, Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.
Baldwin
G S
GS
D015075:2-Aminopurine / Q000378:metabolism; D019943:Amino Acid Substitution; D001483:Base Sequence; D045647:DNA Glycosylases; D005453:Fluorescence; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D009841:Oligonucleotides; D015075:2-Aminopurine; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase