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Nucleic Acids Res. 2019 Jan 30. doi: 10.1093/nar/gkz045. [Epub ahead of print]

Single molecule glycosylase studies with engineered 8-oxoguanine DNA damage sites show functional defects of a MUTYH polyposis variant.

Author information

1
Department of Molecular Physiology and Biophysics, Robert Larner College of Medicine, University of Vermont, Burlington, VT 05405, USA.
2
Department of Microbiology and Molecular Genetics, Robert Larner College of Medicine and College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA.

Abstract

Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a 'wedge' residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.

PMID:
30698731
DOI:
10.1093/nar/gkz045

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