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Abstract MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cofactor in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cofactor reveals that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal cluster and bound substrate DNA tightly, but were completely inactive. We determine the crystal structure of human MUTYH bound to a transition state mimic and this shows that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, support a model in which the cofactor functions to position and activate the catalytic Asp. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cofactor and the base excision site of MUTYH regulate its DNA repair function. 

Abstract The GO DNA repair system protects against GC → TA mutations by finding and removing oxidized guanine. The system is mechanistically well understood but its origins are unknown. We searched metagenomes and abundantly found the genes encoding GO DNA repair at the Lost City Hydrothermal Field (LCHF). We recombinantly expressed the final enzyme in the system to show MutY homologs function to suppress mutations. Microbes at the LCHF thrive without sunlight, fueled by the products of geochemical transformations of seafloor rocks, under conditions believed to resemble a young Earth. High levels of the reductant H₂and low levels of O₂in this environment raise the question, why are resident microbes equipped to repair damage caused by oxidative stress? MutY genes could be assigned to metagenome assembled genomes (MAGs), and thereby associate GO DNA repair with metabolic pathways that generate reactive oxygen, nitrogen and sulfur species. Our results indicate that cell-based life was under evolutionary pressure to cope with oxidized guanine well before O₂levels rose following the great oxidation event. 

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. 

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. 

The GO DNA repair system protects against GC → TA mutations by finding and removing oxidized guanine. The system is mechanistically well understood but its origins are unknown. We searched metagenomes and abundantly found the genes encoding GO DNA repair at the Lost City Hydrothermal Field (LCHF). We recombinantly expressed the final enzyme in the system to show MutY homologs function to suppress mutations. Microbes at the LCHF thrive without sunlight, fueled by the products of geochemical transformations of seafloor rocks, under conditions believed to resemble a young Earth. High levels of the reductant H₂and low levels of O₂in this environment raise the question, why are resident microbes equipped to repair damage caused by oxidative stress? MutY genes could be assigned to metagenome-assembled genomes (MAGs), and thereby associate GO DNA repair with metabolic pathways that generate reactive oxygen, nitrogen and sulfur species. Our results indicate that cell-based life was under evolutionary pressure to cope with oxidized guanine well before O₂levels rose following the great oxidation event.