BG4 antibody can recognize telomeric G-quadruplexes harboring destabilizing base modifications and lesions.

BG4 is a single-chain variable fragment antibody shown to bind various G-quadruplex (GQ) topologies with high affinity and specificity, and to detect GQ in cells, including GQ structures formed within telomeric TTAGGG repeats. Here, we used ELISA and single-molecule pull-down (SiMPull) detection to test how various lengths and GQ destabilizing base modifications in telomeric DNA constructs alter BG4 binding. We observed high-affinity BG4 binding to telomeric GQ independent of telomere length, although three telomeric repeat constructs that cannot form stable intramolecular GQ showed reduced affinity. A single guanine substitution with 8-aza-7-deaza-G, T, A, or C reduced affinity to varying degrees depending on the location and base type, whereas two G substitutions in the telomeric construct dramatically reduced or abolished binding. Substitution with damaged bases 8-oxoguanine and O6-methylguanine failed to prevent BG4 binding although affinity was reduced depending on lesion location. SiMPull combined with FRET revealed that BG4 binding promotes folding of telomeric GQ harboring a G to T substitution or 8-oxoguanine. Atomic force microscopy revealed that BG4 binds telomeric GQ with a 1:1 stoichiometry. Collectively, our data suggest that BG4 can recognize partially folded telomeric GQ structures and promote telomeric GQ stability.

UV irradiation remodels the specificity landscape of transcription factors.

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE).

Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information with fluorescently tagged proteins isolated from nuclear extracts of human cells. We demonstrated the wide applicability of this novel technique on undamaged DNA and three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), and 8-oxoguanine glycosylase 1 (OGG1). We found that PARP1 binding to DNA nicks is altered by tension, and that UV-DDB did not act as an obligate heterodimer of DDB1 and DDB2 on UV-irradiated DNA. UV-DDB bound to UV photoproducts with an average lifetime of 39 seconds (corrected for photobleaching, τc), whereas binding lifetimes to 8-oxoG adducts were < 1 second. Catalytically inactive OGG1 variant K249Q bound oxidative damage 23-fold longer than WT OGG1, at 47 and 2.0 s, respectively. By measuring three fluorescent colors simultaneously, we also characterized the assembly and disassembly kinetics of UV-DDB and OGG1 complexes on DNA. Hence, the SMADNE technique represents a novel, scalable, and universal method to obtain single-molecule mechanistic insights into key protein-DNA interactions in an environment containing physiologically-relevant nuclear proteins.

Chaperones for dancing on chromatin: Role of post-translational modifications in dynamic damage detection hand-offs during nucleotide excision repair.

We highlight a recent study exploring the hand-off of UV damage to several key nucleotide excision repair (NER) proteins in the cascade: UV-DDB, XPC and TFIIH. The delicate dance of DNA repair proteins is choreographed by the dynamic hand-off of DNA damage from one recognition complex to another damage verification protein or set of proteins. These DNA transactions on chromatin are strictly chaperoned by post-translational modifications (PTM). This new study examines the role that ubiquitylation and subsequent DDB2 degradation has during this process. In total, this study suggests an intricate cellular timer mechanism that under normal conditions DDB2 helps recruit and ubiquitylate XPC, stabilizing XPC at damaged sites. If DDB2 persists at damaged sites too long, it is turned over by auto-ubiquitylation and removed from DNA by the action of VCP/p97 for degradation in the 26S proteosome.

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

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.

Single-molecule analysis reveals TDG exhibits multiple modes of linear diffusion to process 5-formylcytosine.

DNA methylation plays a key role in epigenetics, with 60-80% of CpG sites containing 5-methylcytosine. Base excision repair (BER) is suggested to be the main pathway involved in active DNA demethylation. 5-formylctyosine (5fC), an oxidized moiety of methylated cytosine, is recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. TDG binds avidly to abasic sites and is product inhibited. Using single molecule fluorescence experiments, we saw TDG interact with DNA containing 5fC specifically and non-specifically with lifetimes of 72.9 and 7.5 seconds, respectively. These results indicate that TDG cleaves the 5fC and stays bound for an extended time at the generated abasic site. Mean squared displacement analysis and a two color TDG experiment indicate that TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. The catalytically crippled variants, N140A and R275A/L, have a reduced binding lifetime compared to wild type and Mean Squared Displacement (MSD) analysis indicates that R275L/A moves on the DNA with a faster diffusivity. These results indicate that mutating R275, but not N140 interferes with damage recognition by TDG. Our findings give insight into how TDG searches for its lesions in long stretches of undamaged DNA.