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 (εA) 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 εA prior to N-glycosidic bond hydrolysis, which is the rate-limiting step of the reaction. The binding affinity of AlkA for the εA-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 εA 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 εA, 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 εA-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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Taylor EL,O'Brien PJdoi
10.1021/bi501356xsubject
Has Abstractpub_date
2015-01-27 00:00:00pages
898-908issue
3eissn
0006-2960issn
1520-4995journal_volume
54pub_type
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