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Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by AlkA.


ABSTRACT: Escherichia coli 3-methyladenine DNA glycosylase II (AlkA), an adaptive response glycosylase with a broad substrate range, initiates base excision repair by flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond. We used transient and steady state kinetics to determine the minimal mechanism for recognition and excision of 1,N(6)-ethenoadenine (?A) by AlkA. The natural fluorescence of this endogenously produced lesion allowed us to directly monitor the nucleotide flipping step. We found that AlkA rapidly and reversibly binds and flips out ?A prior to N-glycosidic bond hydrolysis, which is the rate-limiting step of the reaction. The binding affinity of AlkA for the ?A-DNA lesion is only 40-fold tighter than for a nonspecific site and 20-fold weaker than for the abasic DNA site. The mechanism of AlkA-catalyzed excision of ?A was compared to that of the human alkyladenine DNA glycosylase (AAG), an independently evolved glycosylase that recognizes many of the same substrates. AlkA and AAG both catalyze N-glycosidic bond hydrolysis to release ?A, and their overall rates of reaction are within 2-fold of each other. Nevertheless, we find dramatic differences in the kinetics and thermodynamics for binding to ?A-DNA. AlkA catalyzes nucleotide flipping an order of magnitude faster than AAG; however, the equilibrium for flipping is almost 3 orders of magnitude more favorable for AAG than for AlkA. These results illustrate how enzymes that perform the same chemistry can use different substrate recognition strategies to effectively repair DNA damage.

SUBMITTER: Taylor EL 

PROVIDER: S-EPMC4310629 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by AlkA.

Taylor Erin L EL   O'Brien Patrick J PJ  

Biochemistry 20150114 3


Escherichia coli 3-methyladenine DNA glycosylase II (AlkA), an adaptive response glycosylase with a broad substrate range, initiates base excision repair by flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond. We used transient and steady state kinetics to determine the minimal mechanism for recognition and excision of 1,N(6)-ethenoadenine (εA) by AlkA. The natural fluorescence of this endogenously produced lesion allowed us to directly monitor the nucleotide flipping s  ...[more]

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