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Effects of Hydrogen Bonding within a Damaged Base Pair on the Activity of Wild Type and DNA-intercalating Mutants of Human Alkyladenine DNA Glycosylase
Human alkyladenine DNA glycosylase âflipsâ damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by âpushingâ it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA...
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Published in: | The Journal of biological chemistry 2002-08, Vol.277 (35), p.31673-31678 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Human alkyladenine DNA glycosylase âflipsâ damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted
into the DNA helix in place of the damaged base and may assist in nucleotide flipping by âpushingâ it. Mutating this DNA-intercalating
Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating
that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine
by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene,
which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme
and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N
6 -ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the
wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base
to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M204475200 |