Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs

Abstract

Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease.

Keywords: 8-Oxoguanine; Bacterial repair assays; Base excision repair; Fe–S clusters; GFP reporter assay; Glycosylase; MUTYH; MutY; Rifampicin resistance.

Figure 1:. Fe-S cluster in MUTYH.

N-terminal fragment crystal structure of Homo sapiens (Hs) MUTYH (PDB 3N5N) depicting the position of MAP variants adjacent the Fe-S cluster (Luncsford, et al., 2010). The N-terminal domain is in blue, the FCL in green, IDC in red, the Fe-S cluster coordinating Cys ligands in orange, and reported MAP variants in purple, where X denotes a nonsense mutation (Out, et al., 2010).

Figure 2:. Schematic representation of the rifampicin resistance assay.

This assay is used to establish the cellular mutation frequency and correlate these mutagenesis events to deficiencies in MUTYH mediated repair. This assay is accomplished through counting the number of colonies resistant to rifampicin (depicted as red plates) in comparison to samples grown on a non-rifampicin containing plate (depicted in brown). The ratio of the median number of resistant colonies to the average number of viable colonies from the corresponding cultures is used to determine the mutation frequency (Garibyan, et al., 2003).

Figure 3:. Schematic representation of the plasmid based bacterial cell assay to assay MutY-mediated OG:A repair.

The restriction enzyme sites for BamHI, PstI and Bmtl are indicated on the pACYC177 (green) plasmid, and the insert duplex carrying the OG:A mispair is shown in pink. The representative agarose gel on the left shows the expected bands formed after Bmtl digestion of the recovered plasmids. To analyze MutY variants, muty^- Ec are transformed with the appropriate vector expressing the mutant enzyme and compared to the results from cells transformed with plasmids expressing the WT enzyme (Brinkmeyer, et al., 2012).

Figure 4:. Schematic representation of the GFP reporter assay. Repair of the OG:A base pair initiated by MUTYH results in GFP expression.

Here, a chemically synthesized OG containing nucleotide is placed opposite A within the coding region of the GFP reporter construct. By default, full GFP expression is interrupted with a stop codon, leading to a 33 amino acid truncated protein product that does not contain the chromophore core needed for fluorescence (Tsien, 1998). However, excision of A and subsequent repair to C causes the complementary mRNA codon to change from UAA to GAA, and translation of this change restores the wild-type amino acid, Glu, allowing expression of full-length wild-type green fluorescent protein.