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 and provides important information on the impact of functional defects that alter affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay more accurately models features of mammalian cells and therefore provides useful information on the cellular repair properties of MUTYH. Taken together, these assays help to provide cellular context to the interaction of MUTYH and its homologs in how they are able to prevent the accumulation of G:C to T:A transversion mutations and a disease phenotype.

Detection of OG:A Lesion Mispairs by MutY Relies on a Single His Residue and the 2-Amino Group of 8-Oxoguanine

“…12 Cellular lesion repair was determined by transformation of a lesioncarrying plasmid into E. coli expressing WT or H296A, or lacking MutY, followed by plasmid extraction and restriction digestion to measure conversion of the lesion to G:C (Figure 2B). 7,8,13 Remarkably, the results with H296A MutY acting on OG:A substrates in vitro and in cells mirrored those with WT MutY acting on 8OI:A substrates. Specifically, the adenine excision rate constant k 2 was decreased 2-fold in both scenarios (Figures 2A, S2).…”

“…Dissociation constants (K D ) for OG:A and 8OI:A, also using 30 bp substrates, were measured using a catalytically inactive E37S MutY, and for WT and H296A MutY using a noncleavable substrate analogue OG:FA (where FA = 2′-deoxyfluoroadenosine) (Figure S3). Cellular lesion repair was determined by transformation of a lesion-carrying plasmid into E. coli expressing WT or H296A, or lacking MutY, followed by plasmid extraction and restriction digestion to measure conversion of the lesion to G:C (Figure B). ,, …”

Unique Hydrogen Bonding of Adenine with the Oxidatively Damaged Base 8-Oxoguanine Enables Specific Recognition and Repair by DNA Glycosylase MutY

“…Notably, with the OG:I lesion bp high levels of conversion to G:C and detection of C:G in sequencing reactions in muty – cells (Table , Figure S10) suggest that this analogue may be processed by an alternative, MutY-independent repair pathway. I is a natural deamination product of A, and its repair is mediated by the glycosylase activity of Mpg and the endonuclease activity of Endo V. − The MutY-mediated repair was quantified as the difference between percent conversion of the OG:Y lesion bp to G:C in plasmids recovered from muty + and muty – cell lines . We defined “overall cellular repair” as the normalized value of this difference.…”

“…The plasmids were amplified and extracted, and then analyzed by restriction digestion and DNA sequencing to determine the distribution of bps at the lesion site (Figure S10). 44 In these assays, OG:A lesion bps within the plasmid DNA are fully repaired in the presence of MutY to the correct G:C bps (>95%), while in the absence of MutY a mixture of G:C and T:A bps are observed at the location of the lesion site (35% G:C, 65% T:A) consistent with equal replication of both bp partners and the expected levels of correct versus mutagenic replication opposite OG. 26,29 In the absence of MutY, the OG:Y bps containing the A analogues P, 2OA, Z3, 2AP, and ADA were processed similarly to A (∼35% G:C) (Table 1).…”

“…I is a natural deamination product of A, and its repair is mediated by the glycosylase activity of Mpg and the endonuclease activity of Endo V. 45−47 The MutY-mediated repair was quantified as the difference between percent conversion of the OG:Y lesion bp to in plasmids recovered from muty + and muty − cell lines. 44 We defined "overall cellular repair" as the normalized value of this difference.…”