Biology subjects

David, S. S.

Publications and source records attributed to David, S. S..

5 recordsLinked to original sources

bioRxiv2026

MUTYH cancer-associated variants within the interdomain connector differentially impact glycosylase activity and cellular DNA repair

The base excision repair (BER) glycosylase MUTYH initiates repair of 8-oxo-7,8-dihydroguanine (OG): adenine (A) mispairs to prevent G to T transversion mutations. Inherited biallelic mutations in MUTYH are correlated with the cancer pre-disposition syndrome MUTYH-associated polyposis (MAP) and contribute to an increased lifetime risk of colorectal cancer. Over 1,000 germline and somatic MUTYH variants have been reported that are associated with MAP and other cancers, but for most the functional impact is unknown. Herein, we examined a subset of cancer-associated variants (CAVs) localized in the interdomain connector (IDC), which links the N-terminal adenine excision and C-terminal OG recognition domains via its zinc linchpin motif and serves as a hub for downstream repair interactions. In vitro assays measuring glycosylase activity, lesion affinity, and AP endonuclease stimulation revealed no substantial defects relative to wild-type MUTYH. In contrast, a newly optimized mammalian cell assay revealed some IDC variants exhibit reduced repair. These results suggest that some variants disrupt steps downstream of adenine excision, whereas others impair lesion recognition and base excision. This work underscores the value of independent functional assays for accurately assessing variant dysfunction and classification. Analysis of MUTYH variants highlights the complexity of the roles of MUTYH in preserving genomic integrity.

biochemistry↗

bioRxiv2026

Structural Basis for Nucleobase Activation by the Adenine DNA Glycosylase MutY

MutY excises adenine (A) from 8-oxo-guanine:adenine (OG:A) lesions in DNA to initiate base excision repair (BER) and thereby prevent mutations. A catalytic Glu, found at position 43 in the enzyme from Geobacillus stearothermophilus (Gs MutY), protonates the nucleobase at N7 to labilize the N-glycosidic bond. The resulting oxocarbenium ion transition state is stabilized by a covalent DNA-enzyme intermediate and resolved by nucleophilic attack to yield the beta-anomer abasic AP site product. The retaining SN1 mechanism for MutY posits deprotonation of the nucleophile by the catalytic Glu. Here we tested kinetic and structural consequences of Glu replacement and found that E43Q and E43S substitution variants were severely impaired, retained measurable activity, but engage the substrate nucleobase in an anti conformation, rotated by 180 {degrees} from the syn conformation seen in previous substrate complexes. The enzyme-generated AP product is observed in its alpha-anomer configuration for these Glu-replacement variants. Comparison with inverting adenine glycosylases that act on RNA or nucleosides shows that MutYs mechanism is uniquely reliant on one catalytic residue for both leaving group and nucleophile activation, a situation that may serve to ensure only rare adenines paired with OG are excised.

biochemistry↗

bioRxiv2025

Structure, dynamics, and processing of 8oxoG:A in the nucleosome

Eukaryotic genomic DNA is packaged into chromatin through a repeating unit known as the nucleosome. In this chromatin environment, DNA is constantly exposed to several sources of DNA damage, such as reactive oxygen species (ROS), which can lead to the formation of 8-oxo-7,8-dihydroguanine (8oxoG). 8oxoG can base pair with cytosine (8oxoG:C) or form a mutagenic base pair with adenine (8oxoG:A), which can lead to single base transversions if left unrepaired. To date, the structure and dynamics of these two possible 8oxoG base pairs in the nucleosome remain unclear. Furthermore, whether MutY homologue (MUTYH) excises 8oxoG:A base pairs in the nucleosome remains elusive. Here using a combination of cryogenic-electron microscopy, molecular dynamics simulations, and biochemistry we determined the structure and dynamics of 8oxoG:C and 8oxoG:A base pairs in the nucleosome and characterize MUTYH activity in nucleosomal DNA. We found that nucleosomal 8oxoG:C forms a stable base pair using its anti conformation, while nucleosomal 8oxoG:A forms a more dynamic base pair using its syn conformation that is unable to be processed by MUTYH. This work provides fundamental insight into the accommodation of oxidative damage in the nucleosome and how this damage contributes to increased mutagenic transversions in nucleosomal compared to linker DNA.

biochemistry↗

bioRxiv2025

Crystal structure of MutYX: A novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-Oxoguanine recognition sphere

The [4Fe-4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe-4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss in two independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup "MutYX". Interestingly, MutYX compensates for the missing [4Fe-4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing -helix and acquisition of the new -helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residue Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible to specificity for OG:A lesions, while Gln201 bridges OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increases activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable.

biochemistry↗

bioRxiv2024

Structure of human MUTYH and functional profiling of cancer-associated variants reveal an allosteric network between its cluster cofactor and active site required for DNA repair

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 revealed that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal center and bound substrate DNA tightly but were completely inactive. We solved the crystal structure of human MUTYH bound to a transition state mimic and this revealed 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, show 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 regulate DNA repair.

biochemistry↗