{'fraction_non_alphanumeric': 0.03555496956512185, 'fraction_numerical': 0.024106181466587558, 'mean_word_length': 5.022763366860773, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
G-quadruplex recognition and remodeling by the FANCJ helicase

Guanine rich nucleic acid sequences can form G-quadruplex (G4) structures that interfere with DNA replication, repair and RNA transcription. The human FANCJ helicase contributes to maintaining genomic integrity by promoting DNA replication through G4-forming DNA regions. Here, we combined single-molecule and ensemble biochemical analysis to show that FANCJ possesses a G4-specific recognition site. Through this interaction, FANCJ targets G4-containing DNA where its helicase and G4-binding activities enable repeated rounds of stepwise G4-unfolding and refolding. In contrast to other G4-remodeling enzymes, FANCJ partially stabilizes the G-quadruplex. This would preserve the substrate for the REV1 translesion DNA synthesis polymerase to incorporate cytosine across from a replication-stalling G-quadruplex. The residues responsible for G-quadruplex recognition also participate in interaction with MLH1 mismatch-repair protein, suggesting that the FANCJ activity supporting replication and its participation in DNA interstrand crosslink repair and/or heteroduplex rejection are mutually exclusive. Our findings not only describe the mechanism by which FANCJ recognizes G-quadruplexes and mediates their stepwise unfolding, but also explain how FANCJ chooses between supporting DNA repair versus promoting DNA replication through G-rich sequences.


INTRODUCTION
G-quadruplexes (G4s) are four-stranded structures formed by guanine-rich nucleic acids. Any single-stranded (ss) DNA sequence containing four stretches of three or more consecutive guanines can fold into a G-quadruplex through Hoogsteen hydrogen bonding between guanines from each run; these interactions are additionally stabilized by monovalent cations such as sodium and potassium (1). G4-forming sequences are highly abundant and fold into stable structures in human cells, with as many as 716 310 unique G-quadruplexes identified within the human genome (2). This staggering number of structures is not formed simultaneously but any that persist can interfere with DNA metabolism. Small molecules that enhance G-quadruplex stability can disrupt DNA replication and RNA transcription by stalling the respective polymerases (3)(4)(5). For these essential cellular processes to continue unperturbed, the help of specialized proteins, many of which are helicases, is needed to unfold G-quadruplexes (6,7).
Helicases are motor proteins that use adenosine triphosphate (ATP) to fuel two important biochemical activities: (i) duplex unwinding, where double-stranded (ds) nucleic acids are separated into the intermediates of DNA replication, recombination and repair, RNA transcription and splicing; (ii) translocation or directional movement along nucleic acids, which can be coupled to the removal of nucleoprotein complexes and the remodeling of unconventional DNA structures (8,9). Many human helicases, including FANCJ (Fanconi Anemia Complementation group J), coordinate these fundamental activities to support multiple genome maintenance pathways (1,7,8,10). FANCJ (or BACH1) is a Superfamily-2 (SF2) helicase that not only facilitates DNA replication through G4-forming sequences, but also participates in homologous recombination (HR) and interstrand DNA crosslink (ICL) repair (4,(11)(12)(13)(14). Defects in FANCJ can lead to Fanconi anemia, a chromosome instability disorder and to increased susceptibility to various cancers (15,16).
FANCJ has 5 →3 directionality and belongs to a group of human XPD-like DNA helicases, which include XPD, RTEL1 and CHLR1 (17). Crystal structures of archaeal XPD revealed that in addition to the canonical SF2 helicase domains (HD1 and HD2) that form the motor core, an iron-sulfur (FeS) cluster-containing domain and an ARCH domain are inserted into HD1 ( Figure 1A) (18)(19)(20). This architecture is shared by all XPD-like helicases. FANCJ, however, has two additional elements that are absent from XPD. The first is the C-terminal domain that, upon phosphorylation on serine 990, binds to the BRCT domain of the BRCA1 tumor suppressor (21,22). The second region is -terminally FLAGtagged FANCJ helicase was produced in HEK293T cells following transient transfection. The bioFANCJ expression vector also contained a biotin acceptor peptide (GLNDIFEAQKIEWHE) at the C-terminus while the biotin-free FANCJ construct lacked this modification. To produce bio-FANCJ, HEK293 cells were co-transfected with pcDNA-BirA. BirA ligase selectively biotinylates the lysine residue within the biotin acceptor peptide. In the cartoon representation of FANCJ, HD1 and HD2 comprise the FANCJ motor core that binds ssDNA and ATP. The FeS containing domain and the ARCH domain are inserted into HD1. The two FANCJspecific features, a C-terminal domain and a modular insertion in HD1, are shown in gray. Coomasie staining and Western blot analysis of purified bio-FANCJ and FANCJ (∼150 kDa) is shown. A full summary including the FANCJ HD and bioFANCJ K141/K142A proteins is provided in Supplementary Figure S1A. (B) TIRFM experiment following the binding of freely diffusing Cy3-labled dT 42 to surface-tethered bioFANCJ. A custom TIRF system was used to generate an evanescent wave for Cy3 illumination. Cy3 emission was collected into an EMCCD camera. A representative dT 42 binding trajectory (green) from a single bioFANCJ is overlaid with an idealized two-state model (black). (C) Dwell-time histogram of the on-times for bioFANCJ-dT 42 binding was fit to a single exponential decay to determine the time constant, ON . The dissociation rate constant for ssDNA binding, k off , was calculated as the inverse of the time constant and was independent of ssDNA concentration (Supplementary Figure S1B). (D) Dwell-time distribution histogram of the off-times for bioFANCJ-ssDNA binding was fit to a single exponential decay to determine the time constant, OFF . The association rate, V on , was calculated as the inverse of the time constant. The association rate constant, k on , was determined by monitoring the dependence of V on on ssDNA concentration (Supplementary Figure S1B). a modular insertion in HD1 that contains a nuclear localization sequence (NLS), as well as a binding site for the MLH1 DNA mismatch repair protein (23,24). These unique features may contribute to different biochemical properties ofy FANCJ and XPD.
FANCJ unwinds both intramolecular and intermolecular G-quadruplexes in vitro whereas Thermoplasma aci-dophilum (Ta) XPD shows no G-quadruplex melting activity; instead, TaXPD prefers to unwind DNA forks and recognizes damaged DNA (5,(25)(26)(27). Notably, another archaeal XPD helicase, Sulfolobus acidocaldarius (Sa) XPD was shown to have a G4 unwinding activity (6). Several other human helicases are also known to unwind G-quadruplexes: another FeS helicase RTEL1; the RecQ-family helicases BLM, WRN and RECQ4; and PIF1 (7,16,28). FANCJ activity is tightly coupled to Gquadruplex maintenance. G4-containing DNA can be efficiently unwound and replicated in the presence of FANCJ, while its depletion sensitizes human cells to G4-stabilizing compounds and causes persistent DNA replication stalling at G-quadruplexes (4). It is possible that one of its specific structural features allows FANCJ to identify and to process G-quadruplex structure. To understand how FANCJ supports DNA replication through G4-containing sequences, we have employed single-molecule Total Internal Reflection Fluorescence Microscopy (TIRFM) and ensemble G4unfolding assays to investigate the mechanism by which FANCJ mediates G4-remodeling.

Buffers and reagents
All solutions were prepared using reagent-grade chemicals and double distilled water that was further purified with a Barnstead GenPure system (Thermo Scientific, Waltham, MA, USA). Buffers and reagents were filtered through a 0.2 m filter after preparation, and all experiments were performed in Buffer H at 25 • C (25 mM HEPES (pH 7.0), 100 mM KCl, 10 mM MgCl 2 , 5% (v/v) glycerol, 5 mM TCEP).

FANCJ expression and purification
BioFANCJ. Full-length human FANCJ helicase was produced and biotinylated in HEK293T cells (25,29,30). Cells were cultured in presence of 5% CO 2 at 37 • C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 1% penicillin and 1% streptomycin. Upon 90% confluence, cells were transferred to reduced serum media containing 10 M biotin and were co-transfected with pcDNA3-FANCJ-cBAP and pcDNA3-BirA plasmids using branched polyethylenimine (PEI) (MW: 25 000; Sigma Aldrich, St. Louis, MO, USA). A 1:3 ratio of total plasmid DNA to PEI was used for transient transfection (30 g pcDNA3-FANCJ-cBAP, 30 g pcDNA3-BirA, 180 g PEI per 175 cm 2 growth area). After 24 h, transfected cells were harvested by trypsinization (0.25% Trypsin-EDTA, Sigma Aldrich), washed with ice-cold DPBS (Thermo Scientific) and the cell pellet was stored at −80 • C until lysis by freezethaw. The cell paste was resuspended in lysis buffer (50 mM HEPES (pH 7.5), 250 mM NaCl, 1 mM EDTA, 0.1% Tween20 and 10% (v/v) glycerol) containing 1 mM PMSF and protease inhibitor cocktail (Roche, San Francisco, CA, USA). The lysate was clarified by centrifugation (20 000 xg, 4 • C) and anti-FLAG magnetic beads (Sigma Aldrich) were added to the supernatant and incubated with rotation at 4 • C for 2 h. The beads were subsequently washed with lysis buffer and bioFANCJ was eluted with Buffer H containing 150 g/ml 3xFLAG peptide (Sigma Aldrich). Purified bio-FANCJ was passed through a Bio-Gel P6 column (Bio-Rad, Hercules, CA, USA) and concentrated with an Amicon Ultra 0.5 ml centrifugal filter (EMD Millipore, Billerica, MA, USA). Protein aliquots were snap frozen in liquid nitrogen and stored at −80 • C until use. BioFANCJ was visualized by coomassie staining and western blot analysis after SDS-PAGE. Biotin incorporation was detected with anti-biotinalkaline phosphatase antibody (Sigma Aldrich) monitoring reaction with ECF substrate (Thermo Scientific) on a ChemiDoc MP imaging system (Bio-Rad). The FLAG epitope was visualized by incubating bioFANCJ with mouse anti-FLAG primary antibody (Sigma Aldrich), followed by anti-mouse-alkaline phosphatase secondary antibody (Sigma Aldrich) and reaction with ECF.
FANCJ, FANCJ HD and bioFANCJ K141/K142A . Nonbiotinylated full-length human FANCJ helicase and the FANCJ helicase domain were produced in a similar way by transfecting HEK293T cells with either pcDNA3-FANCJ or pcDNA3-FANCJ HD plasmids, both of which lack the biotin acceptor peptide sequence. The pcDNA3-FANCJ HD vector was generated by mutating amino acids 889, 890 and 891 simultaneously to stop codons using pcDNA3-FANCJ as a template, thereby terminating FANCJ synthesis after residue 888. To produce bioFANCJ K141/K142A , codons encoding lysine residues 141 and 142 were both mutated to alanine using site directed mutagenesis with pcDNA3-FANCJ-cBAP as a template, and HEK293T cells were co-transfected with pcDNA3-FANCJ K141/K142A -cBAP and pcDNA3-BirA plasmids. Purification of FANCJ, FANCJ HD and bioFANCJ K141/K142A was carried out as described above for bioFANCJ.

DNA substrates
DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). The sequences of the individual oligos and additional modifications are listed in Supplementary Table S1. DNA duplexes were formed by mixing the complementary strands at equimolar ratios in Buffer H, and the resulting mixture was first heated to 95 • C for 10 min and then slowly cooled to 25 • C over 3 h to allow annealing. Fully annealed DNA substrates were gel purified and stored at 4 • C until use.

Ensemble measurements
G-quadruplex unfolding. FANCJ at the indicated concentrations was incubated with 25 nM of a Cy3 labeled Gquadruplex in Buffer H for 5 min at 25 • C to allow the protein-DNA complex to form. G4 unfolding was initiated by adding ATP to 10 mM. Simultaneously with ATP, a ss-DNA trap strand was introduced in excess (200 nM) over the DNA substrate to hybridize with the unwound quadruplex thereby preventing it from reforming. The unfolding reaction was quenched after 5 min at 25 • C with 0.4 M EDTA and 10% SDS, and the products were separated by 15% PAGE. The individual bands were visualized on a Chemi-Doc MP imager (Bio-Rad) and the band intensities were determined using Image Lab software (Bio-Rad).
DNA unwinding. FANCJ helicase, FANCJ HD or bioFANCJ K141/K142A was pre-mixed with 25 nM of a Cy3 labeled DNA fork at the indicated concentrations for 5 min at 25 • C in Buffer H to allow DNA binding. DNA unwinding was initiated by adding ATP to a final concentration of 10 mM and the reaction was quenched after 5 min at 25 • C with 0.4 M EDTA and 10% SDS. The duplex DNA and unwound ssDNA were separated on a 10% polyacrylamide gel, and the individual bands were visualized on a ChemiDoc MP imaging system and the band intensities were quantified using Image Lab software.
DNA binding. FANCJ-DNA binding was monitored by electrophoretic mobility shift assay (EMSA) and by FeS mediated fluorescence quenching. For EMSA experiments, FANCJ, FANCJ HD or bioFANCJ K141/K142A was mixed with 25 nM of a Cy3 labeled G4 at the indicated concentrations for five minutes at 25 • C in Buffer H. Protein-DNA complexes were separated from free DNA on a 4% polyacrylamide gel and the individual bands were visualized on a ChemiDoc MP imaging system and analyzed with Image Lab software. Fluorescence DNA binding experiments were carried out on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) at 25 • C. A total of 40 nM of Cy3 labeled DNA (fork substrate or G-quadruplex) was placed in the cuvette and FANCJ, FANCJ HD or bioFANCJ K141/K142A was titrated into the sample. Cy3 was excited at 520 nm with 10 nm slit widths while Cy3 fluorescence emission was collected at 570 nm with 10 nm slit widths. The relative Cy3 signal change and the extent of fluorescence quenching by the FANCJ FeS cluster was calculated from the following equation, where F 0 is the initial signal from the Cy3 labeled DNA substrate alone and F i is the fluorescence signal upon the ith addition of protein after correcting for dilution.

Single-molecule TIRF microscopy
Single-molecule imaging was performed on a custom built prism-type TIRFM system as described previously (26,(29)(30)(31). Microscope slides and cover slips were prepared as described to sparsely coat the surfaces with biotinylated PEG and neutravidin (26,(29)(30)(31) . Cy3 and Cy5 (where appropriate) fluorescence emission was collected through a water immersion objective and passed through a Cy3/Cy5 dual bandpass filter into an EMCCD camera. Images were recorded at 10 frames per second and amplification gain of 250 without binning. Single-molecule fluorescence trajectories were extracted from the recorded videos as previously described (26,30). Data collected from two independent experiments were pooled together for analysis.

Analysis of single-molecule data
The individual ssDNA and G-quadruplex binding trajectories were fit to a two-state binding model using QuB software (University of Buffalo). The resulting dwell times for the 'on-states' and 'off-states' were binned and plotted as histograms (30). The resulting dwell time distribution histograms were fit to a single-exponential function using Sigmaplot (Systat Software Inc, San Jose, CA, USA). For single-molecule FRET (smFRET) G-quadruplex unfolding measurements, FRET was calculated from the corrected Cy3 donor and Cy5 acceptor fluorescence intensities using the equation below, where I * A and I * D are the background corrected acceptor and donor intensities, and γ is the ratio between the change in acceptor fluorescence intensity to the change in donor fluorescence intensity upon photobleaching trajectories collected from a particular FANCJ construct were globally analyzed with hidden Markov modeling using an empirical Bayesian approach (ebFRET Matlab Suite) to detect subpopulation transitions (32). The ebFRET Matlab library was used to statistically determine the number of FRET states observed during the course of G4 melting, and the mean lower bound was used to evaluate the number of states that best described each time series.

FANCJ recognizes ssDNA and G-quadruplexes
Full-length human FANCJ helicase, with or without a biotin label as depicted in Figure 1A (and Supplementary Figure S1A), was produced in HEK293T cells and purified as described in Materials and Methods. To monitor ssDNA binding to individual biotinylated FANCJ molecules (bio-FANCJ), equilibrium TIRFM experiments were carried out with bioFANCJ immobilized on a neutravidin-coated slide and Cy3-labeled dT 42 in the solution. Because FANCJ is monomeric at low concentration (33), the observed activities of the surface-tethered bioFANCJ should reflect those of FANCJ monomers. The association of the Cy3-dT 42 to the immobilized bioFANCJ and its subsequent dissociation from the helicase resulted in the appearance and disappearance of Cy3 fluorescence intensity. The corresponding trajectories (time-based changes in Cy3 signal at a particular bioFANCJ locus on the slide) collected from individual bio-FANCJ molecules contained multiple ssDNA binding and dissociation events ( Figure 1B). Each trajectory was fit to a two-state model (black line) that described bioFANCJ as either free (background Cy3 fluorescence) or bound to the ssDNA (high Cy3 fluorescence). The idealized data yielded the on-times and off-times for every binding event. Consistent with this simple model, the dwell-time distribution histograms of the on-times and off-times were both welldescribed by a single exponential decay function. The time constant, ON , obtained from analysis of the on-time distribution histogram ( Figure 1C), reflected the average time the ssDNA oligo remained associated with bioFANCJ and was inversely related to the dissociation rate constant, k off . As expected for a unimolecular reaction, k off was independent of Cy3-dT 42 concentration (Supplementary Figure S1B). In contrast, the time constant determined from analysis of the off-time distribution, OFF , measured the average time between successive ssDNA binding events and was related to the on rate, V on ( Figure 1D). Binding experiments were repeated at multiple Cy3-dT 42 concentrations to obtain the association rate constant, k on (Supplementary Figure S1B). The equilibrium dissociation constant, K D (k off /k on ), for the bioFANCJ-dT 42 interaction was estimated to be 1.2 ± 0.3 nM, which is in excellent agreement with previous ensemble measurements (33).
When the same TIRFM experiments were performed in the presence of 1 mM ATP␥ S, a slowly hydrolyzed ATP analog, the bioFANCJ-dT 42 complexes were short lived (Figure 2A), suggesting that ATP␥ S promoted rapid dissociation of the bioFANCJ-dT 42 complex. In the presence of ATP, however, bioFANCJ moved along Cy3-dT 42 with expected 5 →3 directionality. As bioFANCJ approached the Cy3-labeled 3 terminus, we observed a gradual decrease in Cy3 signal as a result of FeS-mediated quenching of Cy3 fluorescence as a function of distance ( Supplementary Figure S2A) (31,34). The translocation trajectories were sensitive to ATP concentration (Supplementary Figure S2B) and non-processive movements producing partial quenching of Cy3 signal were also detected. Together, these TIRFM data indicate that surface-immobilized bioFANCJ were active in both ssDNA binding and translocation.
Next, we used TIRFM to study the binding of a Cy3labeled G-quadruplex to bioFANCJ. We first chose the human telomeric repeat sequence (5 -(TTAGGG) 4 -Cy3-3 ) for the following reasons. This sequence had been extensively characterized biochemically and structurally; furthermore, this sequence formed a stable hybrid antiparallel Gquadruplex under our experimental conditions containing 100 mM KCl (35)(36)(37)(38)(39). Our G4-binding experiments produced two surprising observations. First, in the absence of a flanking ssDNA overhang, bioFANCJ bound to the G-quadruplex directly ( Figure 2B). Second, while ATP␥ S destabilized the bioFANCJ-dT 42 complex, the bioFANCJ-G4 interaction was not influenced by ATP␥ S (Figure 2B and C). We attributed these unexpected results to a potential secondary binding site on FANCJ that can recognize a G-quadruplex structure, distinct from the primary ss-DNA binding pocket within its canonical SF2 helicase core. Kinetic analysis of the bioFANCJ-G4 binding trajectories ( Figure 2D and E) showed that the affinities of bioFANCJ Dwell-time distribution histogram of the on-times for G-quadruplex binding either in the presence (red) or absence (gray) of nucleotide co-factor. The distribution was fit to a single exponential decay function to determine the time constants from which the kinetic parameters were calculated. (E) Dwell-time distribution histogram of the off-times for G-quadruplex binding either in the presence (red) or absence (gray) of nucleotide co-factor. for G4 and dT 42 were similar (K D ≈ 1 nM); however, the individual rate constants determined from the two sets of experiments differed considerably (Supplementary Figure  S1B versus S2C and S2D). This was consistent with different recognition mechanisms for the two substrates, possibly originating from separate DNA binding sites. Alternatively, ssDNA and G4-binding could occur at the same pocket; however, this would require bioFANCJ to unfold the Gquadruplex upon binding and load the newly released DNA onto the conserved ssDNA interaction motifs.

FANCJ bypasses G-quadruplexes and unwinds downstream DNA
To test whether FANCJ can bypass a G-quadruplex, we first used TIRFM to monitor the unwinding of a surfacetethered reference duplex, and then examined if FANCJ can continue beyond an intervening G-quadruplex and unwind a downstream target. FANCJ-catalyzed unwinding of the reference duplex is shown in the representative trajectory in Figure 3A. In the absence of ATP, all trajectories originating from the immobilized Cy3-labeled substrate showed a stable fluorescence signal ( Figure 3A inset). Upon binding to the DNA substrate and in the presence of ATP, FANCJ initiated DNA unwinding by moving with 5 →3 directionality toward the labeled 3 terminus. As FANCJ progressed through the duplex, its FeS cluster gradually quenched Cy3 fluorescence. The resulting unwinding trajectories showed a linear decrease in Cy3 signal as FANCJ unwound this reference substrate followed by a plateau at the near background Cy3 level and then a complete loss of Cy3 fluorescence indicative of the dissociation of the unwound oligo or Cy3 photobleaching. Note that none of the trajectories where the quenching was observed showed the FANCJ dissociation prior to the complete duplex unwinding or prior to the Cy3 photobleaching; i.e. the fluorescence recovery has not been observed. Each time course was fit to a three segmented line, from which the slope of the transition as well as the total duration for each unwinding event were determined, binned and plotted. The slopes of these transitions, which were related to the unwinding rate, were best described by a Gaussian distribution with a mean of 113 ± 26 intensity units per second ( Figure 3B). Based on the total starting Cy3 signal and the duplex length, FANCJ unwinding occurred with an upper bound of ∼3-4 bp/s. Notably, DNA unwinding activity was also achieved when the experiment was carried out with surface-tethered bioFANCJ and biotin-free DNA (Supplementary Figure S3A).
When a G-quadruplex is introduced between the FANCJ loading site and the duplex, FANCJ must gain entrance to the dsDNA before it can unwind the reporter. As seen from an unwinding trajectory ( Figure 3C), the FeS-mediated quenching of Cy3 signal occurred in two discrete phases instead of a single monotonic decrease. The individual trajectories were fit to a five segmented line consisting of three plateau regions 1, 3 and 5, and slopes 2 and 4, which described the rates of the two characteristic transitions. While slope 2 had a broad and complex distribution (Supplementary Figure S3B), slope 4 was described by a simple Gaussian with a mean of 126 ± 39 intensity units per second ( Figure 3D). Interestingly, this value was the same within error as that of the reference duplex. Experiments performed with a G-quadruplex directly adjacent to the dsDNA, without an ssDNA overhang, detected no unwinding activity (Supplementary Figure S3C). This was not due to a lack of DNA binding since surface-immobilized bioFANCJ bound to this substrate (Supplementary Figure S3D). Hence, direct interaction with the G-quadruplex alone was not sufficient for G4-bypass nor DNA unwinding. We conclude from this result that the second observed transition, as indicated by the distribution of slope 4, reflected the unwinding of the downstream duplex while the initial phase (slope 2) contributed to G4-bypass.

FANCJ unfolds G-quadruplex DNA
Next, we monitored the unfolding of a G-quadruplex in an ensemble experiment (5). FANCJ was pre-incubated with a Cy3-labeled quadruplex and G4-unfolding was initiated by adding ATP. An unlabeled 12 nucleotide ssDNA trap (5 -CCCTAACCCTAA-3 ) complementary to the quadruplex sequence was introduced along with ATP to prevent the unfolded DNA substrate from refolding. Since the unfolded G-quadruplex hybridized with the ssDNA trap, the resulting partial duplex showed reduced mobility on a polyacrylamide gel. Neither ATP nor ATP␥ S enabled FANCJmediated unfolding of a G-quadruplex in the absence of an ssDNA overhang ((TTAGGG) 4 -Cy3-3 ) ( Figure 4A). When FANCJ was given the same DNA substrate with a 5 -dT 10 overhang, the G-quadruplex was unfolded in an ATP-dependent reaction ( Figure 4B, Supplementary Figure S4A), suggesting that motor engagement on the ssDNA as well as motor activity were essential for G4-unfolding.
To test whether the FANCJ C-terminal domain was involved in G-quadruplex recognition, we purified a truncated protein consisting of the helicase domain only (residues 1-888) and performed ensemble G4-unfolding experiments with FANCJ HD . FANCJ HD unfolded Gquadruplexes in the presence of ATP only when the DNA substrate contained a 5 -dT 10 tail ( Figure 4A and B) but formed a complex with a substrate lacking the overhang ( Figure 4D). Hence, a G-quadruplex recognition site resided somewhere within FANCJ HD and not the Cterminal domain. Another distinguishing feature of FANCJ is a modular insertion in HD1 that contains a NLS as well as an interaction site for MLH1 (23,24). We purified FANCJ K141/K142A , which was deficient in MLH1 binding but not in DNA unwinding (24). As expected, our purified FANCJ, FANCJ HD and FANCJ K141/K142A retained DNA binding and unwinding activities (Supplementary Figure S4B and S4C). Remarkably, FANCJ K141/K142A did not bind to a G-quadruplex without a 5 -dT 10 overhang while FANCJ and FANCJ HD formed a stable complex with this substrate ( Figure 4D). Therefore, lysine residues K141 and/or K142 formed direct contacts with the Gquadruplex. This observation was unexpected as it suggests that the G-quadruplex and the MLH1 binding are mutually exclusive. FANCJ K141/K142A , however, maintained G4unfolding activity when the DNA substrate possessed an ssDNA overhang for FANCJ loading ( Figure 4C). This showed that although we uncovered a FANCJ-G4 recognition site, this interaction may be used only to target FANCJ to G4-containing DNA regions. G-quadruplex unfolding was enabled purely by the motor activity of the helicase.

G-quadruplex unfolding occurs stepwise in repeating cycles
We performed TIRFM experiments with the DNA substrate shown in Figure 5A to examine conformational transitions within the G-quadruplex during FANCJ-catalyzed G4-unfolding. This DNA substrate was derived from Figure 3B except a Cy3/Cy5 dye pair, which can undergo Förster resonance energy transfer (FRET), was incorporated across the G4-containing gap. When the Gquadruplex was folded, the fluorophores were in close proximity and had an average FRET value of ∼0.65. Stable signals from Cy3 and Cy5 were detected with the surfacetethered DNA alone and when FANCJ was added to this substrate (Supplementary Figure S5A). When FANCJ and ATP were both included, G4-unfolding was seen as anticorrelated changes in Cy3 and Cy5 fluorescence intensities, where an increase in Cy3 signal was accompanied by a concomitant decrease in Cy5 signal ( Figure 5A). Interestingly, the G-quadruplex in each DNA observed was repeatedly unfolded and refolded. We were surprised by this result since FANCJ was able to unwind a duplex downstream of the G-quadruplex ( Figure 3B). However, that experiment was only sensitive to the complete unwinding of the duplex with the Cy3 positioned at the terminus. Indeed when single-molecule unwinding experiments were carried out with a DNA substrate possessing the Cy3 at the FANCJ entrance site, multiple unwinding initiation attempts were observed from the same FANCJ molecule (Supplementary Figure S5B).
An analogous G4-unfolding experiment was performed with bioFANCJ tethered to the slide and freely diffusing DNA ( Figure 5B). Without ATP, the association of the DNA substrate to bioFANCJ and its dissociation from the helicase resulted in the simultaneous appearances and disappearances of Cy3/Cy5 signals and G4-unfolding was not detected (Supplementary Figure S5C). When ATP was included, anti-correlated changes in Cy3 and Cy5 fluorescence intensities were observed. This showed that an individual FANCJ helicase molecule was sufficient to unfold and refold a G-quadruplex. Consistent with the ensemble results, FANCJ HD and bioFANCJ K141/K142A also facilitated G4-unfolding and refolding ( Figure 5C and D). The sm-FRET trajectories were analyzed with hidden Markov modeling using empirical Bayesian methods (32). This approach provided a statistically rigorous determination of the num- ber of discrete FRET states observed over the course of FANCJ-catalyzed G4-unfolding, along with their relative occupancies and associated transitions. These results are summarized in the transition density plots (TDPs) in Figure  6A. The G4-unfolding experiments performed with freely diffusing FANCJ as well as surface-tethered bioFANCJ yielded similar TDPs with four characteristic FRET states with average values of 0.13 (1), 0.32 (2), 0.49 (3) and, 0.64 (4). Therefore, surface immobilization did not adversely influence the G4-unfolding activity of FANCJ. Formation of a parallel G-quadruplex structure was also observed albeit infrequently as the high FRET peak at 0.73 (40,41). The four major FRET states indicated that FANCJ-mediated G4-unfolding proceeded through two intermediates ( Figure  6B). Each of the transitions likely signifies the removal or rebinding of one (transitions between adjacent states) or two (3⇔1 and 4⇔2 transitions) of the G4 strands ( Figure 6C). The TDP of FANCJ HD contained all characteristic states and transitions of the full-length protein, confirming that the C-terminal domain of FANCJ was not involved in processing G-quadruplexes. FANCJ K141/K142A -mediated G4unfolding also showed the same characteristic FRET states; however, the interconversions between the two higher states were severely diminished (G2⇔G4 and G3⇔G4 interconversions). In other words, the fully folded G-quadruplex was not as readily reformed with FANCJ K141/K142A . This was consistent with residues K141 and K142 participating in G4-binding since disrupting these interactions reduced the stability of the quadruplex and hindered its refolding. As expected, TIRFM experiments with FANCJ K141/K142A did not detect binding to a G-quadruplex without an ssDNA overhang (Supplementary Figure S6A). FANCJ K141/K142A retained ssDNA binding activity and the FANCJ K141/K142A -DNA complex showed the same sensitivity to ATP␥ S as the wild-type FANCJ (Supplementary Figure S6B).
We next tested if FANCJ and FANCJ K141/K142A can bind and unfold a G-quadruplex formed by the DNA sequence (GGGT) 4 . This G4 structure is very stable, as it has a melt-ing temperature of over 85 • C, compared to (TTAGGG) 4 that has a T m of 60 • C (42,43). Single-molecule FRET experiments indicate that both FANCJ (Supplementary Figure  S7A) and FANCJ K141/K142A (Supplementary Figure S7B) can repeatedly unfold and refold this high T m substrate, but through different mechanisms when compared to the human telomeric G-quadruplex. Hidden Markov modeling of the FRET transitions for this G-quadruplex shows five major FRET states: 0.13 (1), 0.21 (2), 0.33 (3), 0.43 (4) and 0.61 (5). Although an initial NMR study suggested that (GGGT) 4 forms an antiparallel G-quadruplex, there is increasing recent evidence indicating that this sequence adopts a parallel conformation (44)(45)(46). We assume that states 1 and 5 correspond to the fully unfolded and folded G-quadruplex, respectively, but the presence of an additional intermediate state could indicate the folding and unfolding of a mixture of antiparallel and parallel structures. The TDP obtained from wild-type FANCJ shows interconversions from state 4 to state 1; hence, multiple strands of the G-quadruplex can be unfolded and refolded simultaneously (Supplementary Figure S7A). FANCJ K141/K142A , on the other hand, unfolds this high T m G-quadruplex in a step-wise manner (Supplementary Figure S8A). However, there is an accumulation of states 3 and 4, while states 1, 2 and 5 are rarely observed. This suggests that the complete unfolding and complete refolding of this substrate are both impaired when the G-quadruplex interaction site is mutated in FANCJ. Gel mobility shift experiments using (GGGT) 4 showed that FANCJ retained G-quadruplex binding activity while FANCJ K141/K142A did not bind to this G-quadruplex (Supplementary Figure S8A and S8B). Taken together, the FANCJ G-quadruplex recognition site is dispensable for G-quadruplex unfolding activity unless the helicase encounters a very stable G4 structure, but is necessary to selectively target different G-quadruplexes and to promote their refolding.

DISCUSSION
The vast majority of helicases examined are able to bind and/or unwind G-quadruplex structures in vitro (1). While these observations suggest that G4-unfolding may be a feature shared among helicases, additional information is needed to determine which of these enzymes could have a preference for G4 substrates and participate in G4remodeling in vivo. There is strong experimental evidence that FANCJ directly participates in G4-processing. FANCJ deficiency increases the number of G-quadruplexes formed in human cells; cell lines from Fanconi anemia patients carrying fancj mutations have deletions overlapping G4forming regions (11). Likewise, disruption of the fancj homolog dog-1 in C.elegans, also results in genetic deletions upstream of guanine-rich DNA (47,48). Consistent with these results, FANCJ activity is essential in cellular extracts for efficient DNA replication through G-quadruplexes (4). In FANCJ depleted cells, small molecules that stabilize Gquadruplexes restrict replication fork movements, uncouple leading and lagging strand synthesis, and generate small ssDNA gaps (4,10,49). These studies represent the bestdocumented links between G-quadruplex unfolding in the human genome and the onset of a disease that is connected to helicase malfunctions. Several other human helicases, including WRN, BLM, RECQ4, RTEL1 and PIF1, have been implicated in the unwinding of G-quadruplexes as well, and mutations in these helicases are similarly associated with genome instability and genetic disorders (28,(50)(51)(52)(53). Without a detailed understanding of their G4-targeting and unfolding mechanisms, it is impossible to distinguish whether these helicases are functionally redundant, specific to distinctive types of G4 structures or used in separate cellular pathways.

A molecular hot pocket for G4 recognition
Since helicases are molecular motors, directional movement along the nucleic acid lattice alone (i.e. ssDNA translocase activity) can result in the removal of nucleoprotein complexes and even in the partial unwinding of dsDNA (54). This potentially explains why many helicases exhibit G4-unfolding activity in vitro, especially when the enzymes are included in vast excess over the DNA substrate. Here, we have shown that in addition to the helicase core that binds and translocates along ssDNA with 5 →3 directionality, FANCJ also possesses a G4-recognition site. Notably, the two lysine residues (K141/K142) that are involved in G-quadruplex recognition also interact with the MHL1 DNA repair protein. Having the same structural motif involved in both, the nucleic acid and protein recognition is unprecedented. The positively charged lysine residues in the recognition site can form essential electrostatic interactions with the negatively charged DNA backbone. But why does this binding pocket have a preference for the Gquadruplex? Structural, biochemical and single-molecule studies of the closely related XPD helicase have provided valuable insight to how ssDNA and forked DNA are loaded onto FANCJ as well as how its subdomains are spatially arranged (18)(19)(20)55,56). However, the FANCJ subdomain containing this G-quadruplex binding site is not present in XPD. An extensive search through available structures and sequences of G4-binding proteins revealed that a peptide derived from the RHAU (or DHX36) protein in complex with a G-quadruplex has several similarities to our findings with FANCJ (57). The G4-binding peptide, Rhau18 (SMHPGHLKGREIGMWYAKKQ), adopts an L-shaped structure, in which a proline residue initiates the formation of an ␣-helix that spans between G5 and A17. The positively charged K8, R10 and K18/19 are observed to sit this helix against the G-quadruplex (57). The terminating amino acids, including the two consecutive lysines, form a loop that anchors into the G-quadruplex and locks the ␣-helix in place, while the N-terminal residues are seen pointing away from the DNA, thereby forming an 'L' shape. Lysines 18/19 in Rhau18 are needed for G4-binding, which is analogous to our data with FANCJ K141/K142A . The FANCJ amino acid sequence around K141 and K142 (129-PEKTTLAAKLSAKKQ-143) resembles the Rhau18 peptide and even possesses the same 'AKKQ' anchoring motif. We expect proline 129 in FANCJ also initiates the formation of an ␣-helix that clips onto G4 DNA with a similar mechanism via K131, K137 and K141/142. This is supported by secondary structure prediction analysis of this FANCJ peptide sequence. Notably, this motif is absent in other G4-unwinding DNA helicases, such as Pif1, RTEL1 and the RecQ-family helicases. The discovery of a G4-specific interaction site explains why replication through G4-forming regions relies so strongly on FANCJ, which uses G4-binding along with its motor activity to precisely target the removal of G4 structures.

Mechanism of G-quadruplex unfolding
Having a G4-specific DNA binding pocket implies that FANCJ can also stabilize a folded G-quadruplex. Our sm-FRET results show that FANCJ can undergo repetitive cycles of G4-unfolding and refolding, during which four major FRET states are observed. These states likely represent a fully folded antiparallel G-quadruplex (FRET ≈ 0.64), an unfolded ssDNA (FRET ≈ 0.13) and two intermediates: three-strand (G3; FRET ≈ 0.49) and two-strand (G2; FRET ≈ 0.32) partially folded structures. The strands are peeled from the G-quadruplex one or two at a time in reversible steps as G4⇔G3, G4⇔G2, G3⇔ssDNA and G2⇔ssDNA ( Figure 6). The refolding reaction is presumably aided by direct interactions between FANCJ and the G-quadruplex. This is consistent with our FANCJ K141/142A results, which in contrast with the wild-type protein, showed impaired transitions between the higher FRET states (diminished G2⇔G4 and G3⇔G4 interconversions) as a result of losing its G4-interaction. While stabilizing a G4 seemingly contradicts with its G4-unfolding function, the binding free energy obtained from this interaction provides a potential mechanism for FANCJ to identify the appropriate DNA substrate. Considering a G4-dsDNA junction would be produced during DNA replication as a result of the replicative polymerase stalling at the G4, the interactions between FANCJ and this junction may have important implications to how FANCJ supports DNA replication through G4-forming sequences. While the S. cerevisiae Pif1 helicase also unfolds G-quadruplexes in repeating cycles (53), unlike FANCJ, it has specificity for a ds/ssDNA junction and patrols the nearby ssDNA to remove obstacles such as dsDNA, nucleoprotein complexes and Gquadruplexes (53,58). FANCJ, on the other hand, targets G4-containing ssDNA gaps and stabilizes the folded Gquadruplex during its unfolding-refolding cycles. This key distinction and its mechanistic implications are discussed in the model below.

Model for DNA synthesis through G-quadruplexes
Our finding that K141/K142 are involved in FANCJ-G4 binding is unexpected since these lysine residues also participate in interaction with the MLH1 DNA mismatch repair protein. Based on the Rhau18-G4 structure, FANCJ cannot use its AKKQ motif to bind a G-quadruplex and to form protein-protein interactions with MLH1 simultaneously ( Figure 7A). Consequently, FANCJ function at a G-quadruplex will be incompatible with its activity that involves MLH1, such as facilitating ICL repair and ensuring the fidelity of homologous recombination via heteroduplex rejection (23,59). This exclusion model could explain how FANCJ partitions between its repair activities and in assisting DNA replication through G4-forming sequences. FANCJ possesses a G-quadruplex recognition site within a modular insertion in its HD1. The amino acid sequence at this region is similar to the G-quadruplex recognition peptide seen in the NMR structure of the Rhau18 G4-recognizing peptide (PDB:2N21). The Rhau18 peptide adopts an ␣-helix that sits on top of the G-quadruplex and is followed by a G4anchoring loop formed by an 'AKKQ' motif. This motif is also present in FANCJ at K141/K142. (B) The AKKQ motif in FANCJ can bind to either MLH1 or to a G-quadruplex. Interaction with MLH1 would allow FANCJ to participate in ICL repair as well as heteroduplex rejection to ensure the fidelity of HR, while G4-binding directs FANCJ to support DNA replication through G4-forming sequences. Lagging strand synthesis is stalled when Pol␦ encounters a G-quadruplex. FANCJ is targeted to the resulting G4-ssDNA gap and undergoes G4-unfolding and refolding. This process is repeated until FANCJ recruits a translesion DNA synthesis polymerase such as REV1 either through direct interactions via their respective PIP-RIR sequences or through mediated interactions with PCNA.
Efficient replication of G-quadruplexes was shown to require FANCJ working in concert with either the REV1 translesion DNA synthesis (TLS) polymerase, or the WRN and BLM DNA helicases although it is not known why there are two FANCJ-dependent pathways (60). Since mutations in C. elegans DOG-1, a FANCJ-like helicase, led to defects in lagging strand synthesis (47), we envision that Pol␦ is stalled when it encounters a G-quadruplex in the lagging strand ( Figure 7B). Note, that while the cartoon representing our model is drawn with the lagging strand G4, a similar scenario can be envisioned for the leading strand. FANCJ is then targeted to this DNA substrate via direct interactions with the G-quadruplex and the resulting ssDNA gap at the 5 -side of the block. G4unfolding then proceeds in the 5 →3 direction since it is driven by FANCJ helicase/translocase activity. The FeS domain is thought to facilitate strand separation during DNA unwinding and is positioned in front of the two motor domains (55) while the G-quadruplex is held by the AKKQ motif. Once FANCJ unfolds the last remaining strand of the G-quadruplex, a DNA polymerase can use the newly released ssDNA as a template for synthesis. The dependence on REV1 and FANCJ to maintain epigenetic stability near G-quadruplexes suggests that REV1 may be the DNA polymerase for this reaction (60). Moreover, since REV1 has a specificity for incorporating cytosine, it would efficiently replicate DNA across from the unfolded G-quadruplex (60,61). The partial stabilization of the G-quadruplex as a result of FANCJ binding may be needed to keep FANCJ nearby until REV1 is recruited to the DNA, which may prevent the refolding of G4 DNA structures (62). FANCJ likely recruits REV1 either directly or through interactions with PCNA, since residues 1001-1017 of FANCJ (SWSSFNSLGQYFTGKIP) form a putative PCNA-interacting (PIP) motif, which can also serve as a REV1-interacting (RIR) regions, as shown for other PIP sequences (63). It is likely that after the DNA synthesis across the first few Gs, REV1 hands the substrate over to other TLS or replicative polymerase (62). Our model for Gquadruplex recognition and remodeling by FANCJ is based on the structure formed by the human telomeric repeat sequence and by a more stable (GGGT) 4 G4. The unfolding of other G-quadruplex arrangements formed by different DNA sequences may involve more complex mechanisms and necessitate a division of labor among the G4-unfolding helicases, such as the simultaneous activities of the 5 →3 FANCJ helicase and the 3 →5 WRN or BLM helicases (16,60).