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

Abstract

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.

Damage sensor role of UV-DDB during base excision repair

“…Recombinant human OGG1 protein was purchased from Novus Biologicals or purified from bacterial cells as a GST fusion as previously described 54 . Mouse MUTYH was purified as described previously 55 . Recombinant WT or a catalytically-dead triple mutant (K87E/E96Q/D210N) His-tagged human APE1 was expressed in E. coli and purified as previously described 47 , 56 .…”

The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn2+ Ligands and Roles in Damage Recognition and Repair

“…The following protein purification is based on a previous protocol, 19, 43–44 with the following optimizations. BL21 DE3 (NEB) competent cells co-expressing a pRKISC plasmid were transformed with the Mutyh pET28a plasmid by a 45 second heat shock.…”

“…A 30 bp oligonucleotide (5ʹ - CGATCATGGAGCCACXAGCTCCCGTTACAG - 3ʹ) and its complement, (5ʹ - CTGTAACGGGAGCTYGTGGCTCCATGATCG - 3ʹ) with a central X (8-oxoG) and Y (A or FA) were generated for in vitro studies as previously described. 43 DNA used in fluorescence polarization studies also incorporated a 5’ 6-FAM (6-carboxyfluorescein) on the FA containing strand. Oligonucleotides containing the central 8-oxoG or FA were deprotected and cleaved from the solid support column by incubation in NH 4 OH, with the addition of 2-mercaptoethanol to 8-oxoG samples.…”

Recognition of a tandem lesion by DNA bacterial formamidopyrimidine glycosylases explored combining molecular dynamics and machine learning

“…Besides, other several important MutM residues (K60, H74, Y242, K258, and R264) are known to stabilize the DNA helix by interacting with its backbone [26] . Interestingly, iron-sulfur cluster cofactors can play an important role in lesion processing by repair enzymes [29] including glycosylases (as shown for MutY and its homologs [30] ), highlighting the finely-tuned redox mechanisms which unfortunately require very complex techniques to be modeled.

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