Abstract
The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The beta-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes beta and beta-delta elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.
Highlights
Over the last 15 years, multiple laboratories have investigated the catalytic mechanism of DNA glycosylases that initiate the base excision repair (BER1) pathway
The three new enzyme structures (K20A, K142A, and K20A with adenine) of the 26 kDa catalytic domain of MutY that are reported closely match the X-ray crystal structures of cMutY with either adenine or imidazole bound in the base specificity pocket that were previously solved (Fig. 1A)
These new structures and the original structural data are consistent with localizing the active site of MutY at the inter-domain cleft region and suggest putative amino acid residues that contribute to the glycosylase and lyase chemistry of MutY (Fig. 1B) [12]
Summary
The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7, 8dihydroguanine (8-oxoG) or guanine in duplex DNA. The X-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary β-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35 Å structure of MutY in complex with adenine, that the abasic site (AP) lyase activity is alternatively regulated by two lysines, K142 and K20. The β-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. The final spectrum of DNA products generated, reflect the outcome of a delicate balance of closely related equilibrium constants
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