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

Abstract

How human DNA repair proteins survey the genome for UVinduced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2) 2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.DNA damage recognition | single-molecule tracking | DNA tightrope | human nucleotide excision repair U nrepaired photoproducts in the genome arising from exposure to UV irradiation can be highly mutagenic and three pathways have evolved in mammalian cells to process these lesions, which include (i) global genomic repair, (ii) transcription-coupled repair, and (iii) translesion synthesis (1-5). During global genomic repair, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] are repaired by the nucleotide excision repair (NER) pathway that recognizes and excises bulky helix distorting lesions in the genome (6, 7). The recognition of CPD lesions in UV-damaged chromatin is mediated by UV-damaged DNAbinding protein (UV-DDB), composed of the tightly associated heterodimer of damage-specific DNA binding protein (DDB) 1 (p127) and DDB2 (p48) (5,8). Following surveillance and CPD identification by UV-DDB, NER proceeds via lesion handover to XPC-hHR23B-centrin2 (XPC) followed by damage verification, helix opening and stabilizing of the repair intermediates, dual incision of the DNA in the context of the lesion, repair synthesis, and DNA ligation (7). In contrast to global genomic repair, transcription-coupled repair is initiated when CPD lesions in transcribed chromatin cause stalling of RNA polymerases (3). In mammalian NER, these two pathways converge after damage detection and are orchestrated by over 30 different gene products (9). Deficiencies in the molecular functions in seven of these NER proteins lead to various forms of the autosomal recessive disor...

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process

“…To directly visualize the Rad4-Rad23 search process, we performed single-molecule tightrope assays (Ghodke et al, 2014; Kad et al, 2010) using N-terminally histidine-tagged Rad4 labeled with streptavidin-conjugated quantum-dots (SAQD) through biotinylated anti-histidine-tag antibody (HisAb) (Figure 1B and S1). The wildtype (WT) Rad4-Rad23 used here is essentially the same as that in crystal structures (Figure 1A), spanning all four DNA-interacting domains of Rad4 (His-scRad4 101-632) and all Rad23 domains except for an internal UBA1 domain (Rad23 1-398_Δ135-299).…”

“…We thus characterized the Rad4-Rad23 behavior on DNA substrates that harbor one type of DNA lesion in the same repeating sequence context. To this end, we made long DNA-damage arrays by tandemly ligating multiple linearized plasmids, each contained either one CPD or one fluorescein-modified deoxythymidine (Fl-dT) per 2,030 bp, as previously described (Ghodke et al, 2014). Rad4 binds tightly to Fl-dT (Krasikova et al, 2013), making it a model substrate with high specificity (Figure S2A).…”

“…We used a single-molecule DNA tightrope assay (Kad et al, 2010) and atomic force microscopy (AFM) (Ghodke et al, 2014; Yeh et al, 2012) to (1) directly test the two-stage damage recognition model; (2) visualize how Rad4 searches for DNA damage; and (3) explore the specific role of BHD3. Using different DNA lesions and protein variants, we provide a model for how Rad4 utilizes different structural domains to achieve damage recognition in a dynamic process.…”

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

“…It has been proposed that a conformational switch between a rapidly diffusing "search" mode and a more tightly bound "recognition" mode is needed for a protein to identify a potential target site without losing overall speed (23)(24)(25)(26). Consistent with these arguments, alternative binding modes of various proteins interacting with nonspecific DNA have been observed (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37), and have also been inferred from singlemolecule studies of proteins diffusing on DNA (13,(38)(39)(40). Microsecond-to-millisecond conformational fluctuations have been reported in proteins nonspecifically bound to DNA in a few systems amenable to NMR (28,32,36).…”

Visualizing the search for radiation-damaged DNA bases in real time

“…To accomplish this, we have constructed a plasmid engineered to introduce our lesion of choice and will use a strategy similar to the one described by Gorman et al . for mismatch repair (Gorman et al, 2010) and also used for NER (Ghodke et al, 2014). …”