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

Chandrima Majumdar, Nicole N. Nuñez, Alan G. Raetz, Cindy Khuu, Sheila S. David

Research output: Contribution to journalArticle

1 Scopus citations

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.

Original languageEnglish (US)
JournalMethods in Enzymology
DOIs
StateAccepted/In press - Jan 1 2018

Keywords

  • 8-Oxoguanine
  • Bacterial repair assays
  • Base excision repair
  • Fe-S clusters
  • GFP reporter assay
  • Glycosylase
  • MutY
  • MUTYH
  • Rifampicin resistance

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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