Role of DNA Flexibility in Sequence-Dependent Activity of Uracil DNA Glycosylase

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Abstract
Uracil DNA glycosylase (UDG) is a base excision repair enzyme that specifically recognizes and removes uracil from double- or single-stranded DNA. The efficiency of the enzyme depends on the DNA sequence surrounding the uracil. Crystal structures of UDG in complex with DNA reveal that the DNA is severely bent and distorted in the region of the uracil. This suggests that the sequence-dependent efficiency of the enzyme may be related to the energetic cost of DNA distortion in the process of specific damage recognition. To test this hypothesis, molecular dynamics simulations were performed on two sequences representing extreme cases of UDG efficiency, AUA/TAT (high efficiency) and GUG/CAC (low efficiency). Analysis of the simulations shows that the effective bending force constants are lower for the AUA/TAT sequence, indicating that this sequence is more flexible than the GUG/CAC sequence. Fluorescence lifetimes of the adenine analogue 2-aminopurine (2AP), replacing adenine opposite the uracil, are shorter in the context of the AUA/TAT sequence, indicating more dynamic base−base interaction and greater local flexibility than in the GUG/CAC sequence. Furthermore, the KM of Escherichia coli UDG for the AUA/TAT sequence is 10-fold smaller than that for the GUG/CAC sequence, while the kcat is only 2-fold smaller. This indicates that differences in UDG efficiency largely arise from differences in binding and not catalysis. These results link directly flexibility near the damaged DNA site with the efficiency of DNA repair.