A Zinc Linchpin Motif in the MUTYH Glycosylase Interdomain Connector Is Required for Efficient Repair of DNA Damage

Engstrom, Lisa; Brinkmeyer, Megan K.; Ha, Yang; Raetz, Alan G.; Hedman, Britt; Hodgson, Keith O.; Solomon, Edward I.; David, Sheila S.

doi:10.1021/ja502942d

Cited by 23 publications

(89 citation statements)

References 26 publications

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“…Indeed, the Q324H/R variants helped to illustrate the importance of the IDC in mediating protein-protein interactions that play an important role in DNA repair and the DNA damage response [14, 88, 143]. Similarly, the MAP-associated variant V315M has been shown to weaken a critical 9-1-1 complex binding interaction to MUTYH [146].…”

Section: Mutyh-associated Polyposismentioning

confidence: 99%

“…All but a few recently identified homolog lineages of MutY possess this iron-sulfur cluster [149]. The second cofactor is a recently identified Zn 2+ ion, coordinated to three Cys residues in the interdomain connector (IDC) of higher eukaryotic MutY homologs; this region of MUTYH has been dubbed the “Zn 2+ linchpin motif” to embody its structural and functional roles [14]. These two MUTYH cofactors are essential for efficient repair in cells, but their exact role in DNA repair and regulation is still under investigation [14, 134, 150, 151].…”

Section: Mutyh and Its Metal Cofactorsmentioning

confidence: 99%

“…In this review, we will highlight recent insights into the structural and functional properties of MutY, MUTYH and MAP variants. For example, recent investigations of MAP variants in the David laboratory resulted in the discovery of a second metal binding site in mammalian homologs of MUTYH [14]. Furthermore, despite MutY’s discovery almost 30 years ago, new features of MutY base excision have been revealed prompting revisions to the accepted mechanism for MutY, and implicating that similar revisions may be appropriate for related glycosylases [15].…”

Section: Introduction: Muty’s Nobel Heritagementioning

confidence: 99%

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

Banda

Nuñez

Burnside

et al. 2017

Free Radical Biology and Medicine

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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. 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.

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Section: Mutyh-associated Polyposismentioning

confidence: 99%

Section: Mutyh and Its Metal Cofactorsmentioning

confidence: 99%

Section: Introduction: Muty’s Nobel Heritagementioning

confidence: 99%

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

Banda

Nuñez

Burnside

et al. 2017

Free Radical Biology and Medicine

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“…These condition are also permissive to allow for activity to be observed; notably, however, amino acid variations that significantly alter affinity for the OG:A mismatch and ability to mediate base excision will reduce the active fraction. Thus, the active fraction is a useful gauge of the critical nature of a particular region of MUTYH or amino acid in OG:A recognition and excision [56]. Owing to variation in active enzyme fraction among different preparations of the same variant, we determined the active fraction for several enzyme preparations (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…In the structure of an N-terminal fragment of MUTYH lacking the C-terminal domain, the IDC region forms a “docking scaffold” for interactions with other proteins, such as the 9-1-1 complex [24]. We recently discovered a coordinated Zn(II) ion within the IDC and have shown that MUTYH lacking the Zn(II) ion has low levels of active enzyme [56]. We have dubbed this region a “Zinc-Linchpin Motif” and suggest that Zn(II) coordination in the IDC facilitates the ability of the OG-recognition and catalytic domains to effectively engage the DNA substrate to effect adenine excision.…”

Section: Discussionmentioning

confidence: 99%

Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1

Brinkmeyer

David

2015

DNA Repair

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MUTYH is a base excision repair (BER) protein that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.

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Fe–S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements

Nuñez

Majumdar

Lay

et al. 2018

Methods in Enzymology

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A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S] cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.

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A Zinc Linchpin Motif in the MUTYH Glycosylase Interdomain Connector Is Required for Efficient Repair of DNA Damage

Cited by 23 publications

References 26 publications

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

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

Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1

Fe–S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements

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