Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine

Abstract

Reactive oxygen and nitrogen species (RONS) may infringe on the passing of pristine genetic information by inducing DNA inter- and intra-strand crosslinks, protein-DNA crosslinks, and chemical alterations to the sugar or base moieties of DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent DNA lesions formed by RONS and is repaired through the base excision repair (BER) pathway involving the DNA repair glycosylases OGG1 and MUTYH in eukaryotes. MUTYH removes adenine (A) from 8-oxoG:A mispairs, thus mitigating the potential of G:C to T:A transversion mutations from occurring in the genome. The paramount role of MUTYH in guarding the genome is well established in the etiology of a colorectal cancer predisposition syndrome involving variants of MUTYH, referred to as MUTYH-associated polyposis (MAP). In this review, we highlight recent advances in understanding how MUTYH structure and related function participate in the manifestation of human disease such as MAP. Here we focus on the importance of MUTYH's metal cofactor sites, including a recently discovered "Zinc linchpin" motif, as well as updates to the catalytic mechanism. Finally, we touch on the insight gleaned from studies with MAP-associated MUTYH variants and recent advances in understanding the multifaceted roles of MUTYH in the cell, both in the prevention of mutagenesis and tumorigenesis.

Keywords: 8-oxoguanine; Base excision repair; Fe-S clusters; Glycosylase; MUTYH; MUTYH-associated polyposis; MutY.

Figure 1

The DNA contexts of 8-oxoG. When a normal G:C base pair (A) is damaged to form an 8-oxoG:C base pair (B), 8-oxoG may adopt a syn conformation about its glycosidic bond to form a stable 8-oxoG:A base pair after a round of replication (C). Following a subsequent round of replication, where A is in the template DNA strand, a T:A base pair results (D).

Figure 2

The “GO” repair pathway. Depiction of the GO repair pathway, featuring the key base excision responsibilities of OGG1 and MUTYH.

Figure 3

Structural insight into the mechanism of MutY. (A) Crystal structure of prokaryotic Gs MutY (PDB ID: 5DPK) bound to DNA (grey) containing 8-oxoG (hot pink) opposite the azasugar transition state mimic, 1N (yellow). The N-terminal domain of MutY (blue), contains the catalytic machinery for adenine excision, including the signature HhH-GPD motif (purple) and the [4Fe-4S]²⁺ cluster (orange and yellow spheres). The NUDT1-like C-terminal domain of MutY (red) primarily serves in 8-oxoG recognition. The IDC (green) of bacterial homologs of MutY wraps across the DNA major groove. (B) Close up of 8-oxoG (hot pink) in the active site of MutY. 8-oxoG experiences extensive interactions with MutY, stabilizing the damaged base (interacting amino acids are labeled and colored in forest green). Comparison of the substrate base analogs (C) FA (of PDB ID: 3G0Q, FLRC crystal structure) and (D) 1N (of PDB ID: 5DPK, TSAC crystal structure) within the active site of Gs MutY. Amino acid residues of MutY that are in close proximity to the FA/1N (yellow) nucleotides are labeled and colored in forest green. The position of the potential catalytic water molecule within close proximity to N1’ of 1N is labeled as H₂O in panel D. In panels B, C, D, water molecules are depicted as red spheres, distances are demonstrated by yellow dashes, and elements are depicted with oxygen in red, nitrogen in blue, and phosphorous in orange.

Figure 4

Revised mechanism for MutY-catalyzed adenine excision. (A) The recently proposed double displacement mechanism for MutY enzymes based on methanolysis studies that revealed retention of configuration at the anomeric carbon. This result suggests the involvement of a covalent intermediate, most likely involving Asp 144, in analogy to similar work with retaining O-glycosidases. (B) The oxacarbenium ion-like transition states formed during the proposed MutY mechanism. For both TSs, a highly dissociative transition state based on previous KIE measurements is shown; the extent of participation of the nucleophile in the two TSs remains to be established.

Figure 5

MAP map. Crystal structure of prokaryotic Gs MutY (PDB ID: 5DPK) bound to DNA (light grey) containing 8-oxoG (black) opposite 1N (yellow). MAP variants and domains discussed in this review are indicated on the MAP map and correspond to the MUTYH homologous positioning on the structure of MutY. *Note, for consistency with previous work, the amino acid positions are based on the 535 amino acid MUTYH isoform α-3 (UniProt ID# Q9UIF7-3). The positions of MUTYH MAP variants in the LOVD are categorized both by the longer 549 MUTYH isoform numbering (such that Y165C corresponds to Y179C), as well as the numbering of the shorter aforementioned isoform.

Figure 6

The fragment crystal structure of H. sapien MUTYH (PDB ID: 3N5N) lacking the C-terminal 8-oxoG recognition domain, with close-ups to metal binding sites. Located in the N-terminal catalytic domain of MUTYH (blue) is the [4Fe-4S]²⁺ cluster (Fe^2+/3+ in orange spheres and sulfide in yellow spheres, with Cys ligands as sticks, sulfur highlighted also in yellow) as well as the HhH GPD motif (purple). Linking the N- and C-terminal domains is the IDC (green), which contains the Zn²⁺ linchpin motif (Cys ligands responsible for coordinating Zn²⁺ metal shown as sticks, sulfur highlighted in yellow) and is conserved amongst higher eukaryotic homologs.

Figure 7

Schematic illustrating the roles of MUTYH in preventing carcinogenesis. MUTYH activity prevents of mutations associated with 8-oxoG by removing A from 8-oxoG:A mismatches (blue). However, under conditions that produce overwhelming numbers of 8-oxoG:A lesions, the A excision activity of MUTYH, and subsequent product of strand breaks provides signals that lead to cell death that prevent tumorigenesis (green).