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Abstract DNA glycosylase MutY plays a critical role in suppression of mutations resulted from oxidative damage, as highlighted by cancer-association of the human enzyme. MutY requires a highly conserved catalytic Asp residue for excision of adenines misinserted opposite 8-oxo-7,8-dihydroguanine (OG). A nearby Asn residue hydrogen bonds to the catalytic Asp in structures of MutY and its mutation to Ser is an inherited variant in human MUTYH associated with colorectal cancer. We captured structural snapshots of N146S Geobacillus stearothermophilus MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site products to provide insight into the base excision mechanism of MutY and the role of Asn. Surprisingly, despite the ability of N146S to excise adenine and purine (P) in vitro, albeit at slow rates, N146S-OG:P complex showed a calcium coordinated to the purine base altering its conformation to inhibit hydrolysis. We obtained crystal structures of N146S Gs MutY bound to its abasic site product by removing the calcium from crystals of N146S-OG:P complex to initiate catalysis in crystallo or by crystallization in the absence of calcium. The product structures of N146S feature enzyme-generated β-anomer abasic sites that support a retaining mechanism for MutY-catalyzed base excision.
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This content will become publicly available on August 18, 2026
Structures and activity for Geobacillus stearothermophilus MutY Glu43 substitution variants: Implications for evolution of an adenine DNA glycosylase
Background: MutY initiated base excision repair (BER) protects against mutations that otherwise result from oxidative damage to guanine. The 8-oxo-7,8-dihydro-guanine lesion (OG) pairs equally well with C or A, a source of ambiguity that explains high rates of GC → TA mutations for biallelic defects in the gene encoding MUTYH in humans. MutY/MUTYH intercepts the A:OG lesion and removes the chemically correct but informationally defective adenine nucleobase. A catalytic Glu – Glu43 in Geobacillus stearothermophilus MutY (Gs MutY); Glu37 in Escherichia coli MutY (Ec MutY); Glu134 in human MUTYH – is a defining chemical motif for A:OG adenine DNA glycosylases that is not found in other helix-hairpin-helix family members. While the importance of Glu for MutY’s catalytic mechanism is well understood, its origin is unknown. Discovery: Here we tested the structural and kinetic consequences of Glu replacement and found an alternate, slower mechanism, that yields a different stereoisomer for the AP site product. The impact on catalytic rate was consistent removal of acid/base catalysis and retention of transition state stabilization. High-resolution structures for E43Q and E43S substitution variants of Gs MutY show substrate disengagement and the enzyme-generated AP product in its alpha-anomer configuration. These results suggest that acquisition of Glu was a key innovation for emergence of the MutY/MUTYH lineage from an ancestral DNA glycosylase.
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- Award ID(s):
- 2204229
- PAR ID:
- 10654088
- Publisher / Repository:
- ACS SciMeetings
- Date Published:
- Format(s):
- Medium: X
- Institution:
- University of Utah
- Sponsoring Org:
- National Science Foundation
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