Substrate recognition by Escherichia coli MutY using substrate analogs

Abstract

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

“…30 MutY exhibits particularly high affinities for duplexes containing OG:FA bps (K d = 0.8 ± 0.4 nM). 25,31 The extents of conversion of OG:FA to G:C was significantly less than that observed with OG:A and OG:Z3 in the presence of MutY (57 ± 2 %). The G:FA mismatch was converted to G:C (44 ± 1 %) at a level that was similar to G:A and G:Z3 in the presence of MutY (Fig.…”

“…17 For E37S MutY, binding titrations were first performed to determine the amount of "active" enzyme. 18 Glycosylase activity assays and dissociation constants (K d ) were measured as described previously 17,31,39 . Buffer conditions for the glycosylase assays were 20 mM Tris-HCl pH 7.6, 10 mM EDTA, 0.1 mg/mL BSA, and 30 mM or 150 mM NaCl.…”

Adenine Release Is Fast in MutY-catalyzed Hydrolysis of G:A and 8-Oxo-G:A DNA Mismatches

“…The steady state rate constant is much lower, 0.003 min Ϫ1 with OG:A and 0.02 min Ϫ1 for G:A, which has been attributed to rate-limiting AP-DNA release (9). The steady state rates parallel the higher affinity of MutY for OG:AP sites than for G:AP sites, with dissociation constants (K d ) of 50 -120 pM and 2-21 nM, respectively (11)(12)(13). It was not known whether this reflects slow adenine release followed by rapid AP-DNA release, or vice versa, or coordinated release of both.…”

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…MutY, structurally similar to Endo III [18][19][20][21], is another BER glycosylase that contains a [4Fe-4S] cluster [20]. However, MutY instead removes adenine from 8-oxo-guanine:adenine mispairs [22][23][24][25][26][27][28][29][30][31][32][33][34].…”