Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway.
Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.
Division of Molecular Biosciences, Sir Alexander Fleming Building, Chemical Biology Centre, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Seden
S
Grippon
Qiyuan
Q
Zhao
Tom
T
Robinson
Jacqueline J T
JJ
Marshall
Rory J
RJ
O'Neill
Hugh
H
Manning
Gordon
G
Kennedy
Christopher
C
Dunsby
Mark
M
Neil
Stephen E
SE
Halford
Paul M W
PM
French
Geoff S
GS
Baldwin
D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization; D011485:Protein Binding; D012965:Sodium Chloride / Q000737:chemistry; D044502:Thymine DNA Glycosylase / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012965:Sodium Chloride; D004247:DNA; D044502:Thymine DNA Glycosylase; D051981:Uracil-DNA Glycosidase; C113921:mismatch-specific thymine uracil-DNA glycosylase