Title: Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover.
Authors: Jang, Sunbok; Schaich, Matthew A; Khuu, Cindy; Schnable, Brittani L; Majumdar, Chandrima; Watkins, Simon C; David, Sheila S; Van Houten, Bennett
Published In Nucleic Acids Res, (2021 08 20)
Abstract: 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.
PubMed ID: 34232996
MeSH Terms: Adenine/chemistry; Animals; DNA Damage/drug effects*; DNA Damage/radiation effects; DNA Glycosylases/genetics*; DNA Repair/drug effects; DNA Repair/radiation effects; DNA Replication/drug effects; DNA Replication/radiation effects; Guanine/analogs & derivatives*; Guanine/chemistry; Guanine/pharmacology; Guanine/toxicity; Hydrocarbons, Chlorinated/pharmacology; Hydrocarbons, Chlorinated/toxicity; Mice; Oxidative Stress/drug effects*; Oxidative Stress/radiation effects; Single Molecule Imaging