Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover

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.

Roles for the 8-Oxoguanine DNA Repair System in Protecting Telomeres From Oxidative Stress

“…However, H 2 O 2 and superoxide lead to multiple DNA lesion types, DSBs and SSBs, making it difficult to determine which damage recruits SIRT6. A similar damage sensor and nucleosome remodeling role has also been proposed for UV-DDB, which is recruited to telomeres upon targeted production of 8-oxoG with the FAP-TRF1 system, and stimulates MUTYH activity and turnover (Jang et al, 2019;Jang et al, 2021). It is not clear whether MUTYH recruitment is dependent on replication, particularly since MUTYH can bind 8-oxoG:C base pairs, although in an unproductive manner.…”

Searching for DNA Damage: Insights From Single Molecule Analysis

“…The crosstalk between UV-DDB and another mammalian glycosylase, MUTYH, was also recently studied by our group working in collaboration with the David laboratory at the singlemolecule level (Jang et al, 2021). UV-DDB was shown to stimulate the enzymatic turnover of MUTYH and facilitate the dissociation of MUTYH via transient co-complex formation.…”

“…These interaction networks are complex, and the exact biological importance of pathway crosstalk is still under investigation. However, various proteins in both pathways are involved in the crosstalk between pathways, including the NER proteins UV-DDB, XPC-RAD23B, XPA, CSB and XPG, and BER enzymes OGG1, NTHL1, APE1, and MUTYH (Kumar et al, 2020;Jang et al, 2021). Using singlemolecule fluorescence assays, direct molecular evidence for the importance of how pathway crosstalk contributes to the detection of DNA damage has recently begun to emerge.…”

Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)

“…This latter subunit engages DNA at the site of damage 38 . UV-DDB detects UV-induced photoproducts with high affinity 39 , and the purified protein has been extensively characterized at the single-molecule level for various DNA substrates 30,38,40 . Thus, these previous studies serve as a benchmark in which to validate the behavior of UV-DDB by SMADNE.…”

“…Using the LUMICKS C-trap combined optical tweezers, microfluidics, and 3-color confocal microscope, we precisely define the positions of fluorescently-tagged DNA repair proteins on 48.5 kb DNA substates containing defined types of damage. As shown below, SMADNE provides binding specificity and diffusivity measurements including characterizing multiple proteins simultaneously binding DNA damage with over 4 orders of magnitude of duration (0.1 to >100 s) and a wide range of 1D diffusivity values (from 0.001 to 1 μm 2 s -1 ), with similar precision as other single molecule techniques 4,[26][27][28][29][30][31][32][33][34] . At the same time, SMADNE bridges the complex milieu of the nuclear environment containing thousands of proteins to a system where fluorescently tagged single particles can be followed and characterized.…”