Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA

Abstract

Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix–hairpin–helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe111 insertion.

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

“…32, and single-molecule methods have been used to experimentally validate and visualize various protein search strategies (27,28,(33)(34)(35). We have previously developed a DNA tightrope assay that enables the direct visualization of dynamics of QD-conjugated proteins on DNA (27)(28)(29)(30). Briefly, in this assay, λ-DNA tightropes are strung-up between 5-μm poly-L-lysinecoated beads, which are deposited on a PEGylated coverslip ( Fig.…”

“…These nanoparticles provide superior brightness and resistance to photobleaching compared with conventional fluorophores and fluorescent proteins (36,37). To that end, we systematically tested three strategies for conjugating QDs to UV-DDB and proceeded with a His-Ab conjugation strategy that enabled us to conjugate UV-DDB to SA-QDs using a biotinylated pentaHis antibody, while retaining DNA damage binding activity (SI Materials and Methods, section 1.2.3) (29). We incubated QD UV-DDB with undamaged λ-DNA tightropes or λ-DNA containing on average one lesion per 2,200 bp (SI Materials and Methods, section 2) and observed these interactions in a time window of 900 s. Imaging was performed in the absence of YOYO-1 dye to minimize potential double-strand breaks in the DNA tightropes (SI Materials and Methods, section 1.2.4) (38).…”

“…The QD conjugation strategy described in ref. 29 was used to conjugate UV-DDB (His-DDB1/DDB2) to the penta-His-biotinylated conjugated (Qiagen; His-Ab). SA-QDs (Invitrogen) were conjugated to penta-His-biotinylated conjugated (Qiagen) in a molar ratio of 1:5 for 20 min at room temperature (RT).…”

“…6E). The subdiffusive nature of the UV-DDB (K244E) sliding indicates that this complex performs a constrained Brownian walk on the DNA, suggesting that although mutant DDB2 cannot bind to DNA damage to form persistent complexes, it has several strong interactions with the DNA, such as helix probing with the FQH motif (29). This sliding state may arise from either a conformational change in the monomer of UV-DDB (K244E) that is capable of sliding, or from dimeric UV-DDB (K244E) that fails to stably engage the lesion.…”

“…To better understand damage recognition by UV-DDB, we used a single-molecule DNA tightrope assay (27)(28)(29)(30) to observe the real time interactions of quantum dot (QD)-conjugated wildtype (WT) UV-DDB or UV-DDB containing the K244E mutation in DDB2, with damaged DNA substrates with high temporal and spatial resolution. Observations of individual molecules reveal the presence of short-lived intermediates and heterogeneity in molecular properties that may be lost due to bulk averaging of the properties of an unsynchronized ensemble of molecules.…”