Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover
| Autor: | Bennett Van Houten, Sheila S. David, Chandrima Majumdar, Cindy Khuu, Matthew A. Schaich, Brittani L Schnable, Sunbok Jang, Simon C. Watkins |
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| Rok vydání: | 2021 |
| Předmět: |
DNA Replication
Guanine DNA Repair DNA polymerase AcademicSubjects/SCI00010 1.1 Normal biological development and functioning Genome Integrity Repair and Replication DNA Glycosylases Narese/4 03 medical and health sciences chemistry.chemical_compound Narese/16 Mice 0302 clinical medicine MUTYH Underpinning research Information and Computing Sciences Hydrocarbons Chlorinated Genetics Animals AP site Electrophoretic mobility shift assay heterocyclic compounds 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology Adenine Base excision repair Biological Sciences Molecular biology Hydrocarbons Single Molecule Imaging Chlorinated Oxidative Stress Enzyme chemistry DNA glycosylase 030220 oncology & carcinogenesis biology.protein Generic health relevance DNA Environmental Sciences DNA Damage Developmental Biology |
| Zdroj: | Nucleic acids research, vol 49, iss 14 Nucleic Acids Research |
| Popis: | The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair. |
| Databáze: | OpenAIRE |
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