Yuan Teng

Ting-Ting Wang

Hai-Lei Guo

Ben-Ge Xin

Bo Sun

Shuo-Xing Dou

Xu-Guang Xi

Xi-Miao Hou
email: houximiao@nwsuaf.edu.cn

Fang-Yuan Teng

The HRDC domain oppositely modulates the

Keywords: DNA repair, E. coli, RecQ, G-quadruplex, Helicase, Unwinding, Single-molecule

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genome integrity maintenance by processing a wide variety of DNA structures generated during DNA replication, repair, and recombination [START_REF] Brosh | DNA helicases involved in DNA repair and their roles in cancer[END_REF][START_REF] Vindigni | Probing the structural basis of RecQ helicase function[END_REF][START_REF] Croteau | Human RecQ Helicases in DNA Repair, Recombination, and Replication[END_REF]. These proteins are conserved in both prokaryotes and eukaryotes [START_REF] Larsen | RecQ Helicases: Conserved Guardians of Genomic Integrity[END_REF]. In humans, there are five RecQ helicases: RecQ1, BLM, WRN, RecQ4, and RecQ5. Importantly, mutations in BLM, WRN, and RecQ4 genes cause Bloom, Werner, and Rothmund-Thompson syndromes, which are linked to profound developmental abnormalities and increased cancer risk [START_REF] Mohaghegh | The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases[END_REF]. Meanwhile, the latter two syndromes are also characterized by premature aging. In E. coli, RecQ functions in the RecF recombination pathway to repair the single-stranded DNA (ssDNA) gaps and double-stranded DNA (dsDNA) breaks [START_REF] Handa | Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli[END_REF]. E. coli RecQ is also involved in suppressing illegitimate recombination [START_REF] Hanada | RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli[END_REF], repairing stalled replication forks, and promoting the induction of SOS response [START_REF] Hishida | Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks[END_REF].

The ability of RecQ family helicases to resolve complex DNA structures is associated with their architecture consisting of evolutionarily conserved domains [START_REF] Vindigni | RecQ helicases: multiple structures for multiple functions?[END_REF]. First, a conserved helicase core is formed by two RecA domains that harbor the ATPase cleft and drive the 3′-5′ directed translocation on ssDNA. Second, the helicase core is followed by a RecQ C-terminal (RQC) domain, which contains a Zn 2+ binding domain and a β-hairpin winged-helix domain. RQC is primarily responsible for substrate recognition and DNA unwinding. Besides, an auxiliary element, the helicase, and RNaseD C-terminal (HRDC) domain, which is connected to RQC by a flexible linker, exists in some RecQ helicases, including E. coli RecQ, human BLM, and WRN. The primary sequences and surface properties of the HRDC domain vary remarkably among different species, and HRDC is even absent in two human RecQ helicases [START_REF] Bernstein | Conferring substrate specificity to DNA helicases: role of the RecQ HRDC domain[END_REF][START_REF] Kim | Structure and function of the regulatory HRDC domain from human Bloom syndrome protein[END_REF][START_REF] Newman | Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes[END_REF][START_REF] Sato | Solution structure of the HRDC domain of human Bloom syndrome protein BLM[END_REF][START_REF] Kitano | Crystal structure of the HRDC domain of human Werner syndrome protein, WRN[END_REF][START_REF] Liu | The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins[END_REF]. These evidences then raise important questions about the functions of HRDC in the various DNA transactions of RecQ family helicases, such as translocating on ssDNA, unwinding dsDNA, and resolving G-quadruplex (G4).

In recent years, the duplex DNA unwinding activities of RecQ family helicases have been widely investigated. Single-molecule studies showed that human BLM unwinds duplex DNA in a highly repetitive fashion by switching between unwinding and rewinding modes [START_REF] Yodh | BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation[END_REF][START_REF] Wang | Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase[END_REF]. Similar phenomena have also been reported in other RecQ helicases, such as human or chicken WRN [START_REF] Wu | Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding[END_REF][START_REF] Lee | Multiple RPAs make WRN syndrome protein a superhelicase[END_REF], A. thaliana RecQ2 [START_REF] Klaue | Fork sensing and strand switching control antagonistic activities of RecQ helicases[END_REF], C. elegans HIM-6 [START_REF] Choi | Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase[END_REF], implying that these helicases may use a complex mechanism to unwind duplex DNA rather than the simple unidirectional strand separation. Recently, a very complex dsDNA unwinding behavior with frequent pause, shuttling [START_REF] Harami | Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination[END_REF], and co-existence of two unwinding modes [START_REF] Bagchi | Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB[END_REF] has been reported for E. coli RecQ using magnetic tweezers. Besides, HRDC was shown to suppress the rate of DNA-activated ATPase activity in E. coli RecQ [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF]. However, how E. coli RecQ unwinds duplex DNA when there is no external force, and particularly, the modulation mechanism of HRDC needs further investigation.

In addition to processing duplex DNA, bubble DNA, displacement loops (D-loops), and Holliday junctions, helicases in the RecQ family, such as E. coli RecQ, C. sakazakii RecQ, yeast sgs1, human BLM, and WRN, also participate in resolving G4 DNA structure [START_REF] Voter | A guanine-flipping and sequestration mechanism for G-quadruplex unwinding by RecQ helicases[END_REF]. As non-canonical nucleic acid structures, G4s can be formed in guanine-rich DNA regions and are implicated in several critical cellular processes, including genomic DNA recombination, replication, and telomere maintenance [START_REF] Bochman | DNA secondary structures: stability and function of G-quadruplex structures[END_REF]. Both BLM and WRN helicases operate on a wide range of G4 structures with repetitive cycles of unfolding and refolding [START_REF] Wu | Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding[END_REF][START_REF] Tippana | Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases[END_REF][START_REF] Chatterjee | Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures[END_REF][START_REF] Wu | BLM unfolds G-quadruplexes in different structural environments through different mechanisms[END_REF]. Recently, the widespread existence and potential regulatory roles of G4s have been reported in the E. coli genome [START_REF] Kaplan | G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch[END_REF]. However, the detailed mechanism of how E. coli RecQ acts on G4 structures, and particularly, how HRDC impacts the G4 unwinding activity of RecQ remains to be determined.

In this study, we characterized the activity of E. coli RecQ on different DNA substrates, including 3′-, 5′-partial duplexes, fork DNA, and G4 DNA, as well as the roles of HRDC by single-molecule FRET (fluorescence resonance efficiency transfer). Our results indicate that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with moderate processivity. RecQ is also able to periodically patrol on the ssDNA in 5′-partial duplex, extruding DNA loops. Besides, RecQ unfolds G4 structure in a stepwise and repetitive fashion and maintains G4 DNA in an unfolded state for a long time. More importantly, we discovered that HRDC oppositely modulates the unwinding or patrolling activity of RecQ on duplex DNA (suppressing) and G4 structure (enhancing). Based on these results, we proposed models to explain the different modulation mechanism of HRDC in different DNA transactions.

RESULTS

The atomic structures of the E. coli RecQ catalytic core [START_REF] Bernstein | High-resolution structure of the E.coli RecQ helicase catalytic core[END_REF], C. sakazakii RecQ catalytic core bound to DNA [START_REF] Manthei | Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases[END_REF], and human BLM bound to DNA with HRDC [START_REF] Newman | Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes[END_REF][START_REF] Swan | Structure of human Bloom's syndrome helicase in complex with ADP and duplex DNA[END_REF] were previously resolved (Figure 1A). The HRDC domain is missing in both RecQ structures; however, in BLM, it folds back onto the core and interacts with both RecA domains [START_REF] Newman | Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes[END_REF][START_REF] Swan | Structure of human Bloom's syndrome helicase in complex with ADP and duplex DNA[END_REF], suggesting that E. coli and C. sakazakii RecQ may undergo similar interactions. To comprehensively address the role of HRDC in the enzymatic activity of RecQ, in this study, we examined and compared the activities of wild-type E. coli RecQ (referred to as RecQ herein), and RecQ 523 which lacks the HRDC domain on a series of DNA substrates including 3′-, 5′partial duplexes, fork DNA, and G4 DNA. RecQ Y555A which abolished the ssDNA binding ability of HRDC with a Y555A mutation was also used to further dissect the impact of the interaction between HRDC and ssDNA (Figure 1B) [START_REF] Bernstein | Conferring substrate specificity to DNA helicases: role of the RecQ HRDC domain[END_REF].

HRDC dynamically interacts with the RecA core and ssDNA overhangs, and significantly reinforces RecQ binding on DNA. Before delving into the specific unwinding mechanism of RecQ, we first investigated its DNA-binding activity and the influences of HRDC at the single-molecule level. The substrate (referred to as 16bp 12nt-1) contained a 12-nt ssDNA at the 3′ end of a 16-bp duplex and was anchored onto coverslip by the biotin-streptavidin link (Figure 1C). Cy3 and Cy5 were labeled at the end of the 3′-ssDNA and the 4 th nucleotide inside the duplex. 16bp 12nt-1 displayed stable FRET efficiency at ~0.92 (Figure S1A, Figure 1E). Upon the addition of RecQ, the FRET trace oscillated frequently between ~E0.9 and ~E0.55 in abrupt steps (Figure S1B; Figure 1D, upper panel; Figure 1E;), reflecting the repetitive association and dissociation of RecQ.

HRDC is connected to the remaining portion of RecQ through a long and flexible loop, raising the possibility of dynamic interactions with the RecA core and/or the ssDNA regions of the DNA substrate. In our previous report, we had directly labeled the HRDC domain in RecQ by a Cy5 (referred to as Cy5-RecQ) and examined its interaction with different DNA substrates by smFRET [START_REF] Teng | A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay[END_REF]. We found that the Cy5 labeled HRDC domain can directly interact with the helicase core (Figure S2A-B), the 5′-overhang in fork DNA (Figure S2C), and the free 3′-ssDNA beyond the helicase core (Figure S2D) [START_REF] Teng | A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay[END_REF].

We next addressed the influence of HRDC on the DNA-binding affinity of RecQ. The equilibrium DNA binding assay shown in Figure S3 indicates that, although HRDC itself can negligibly associate with the partial duplex, the deletion of HRDC significantly attenuated in the binding affinity of RecQ, with the KD value increasing from 16.1 ± 0.4 nM to 91.6 ± 5.8 nM. RecQ Y555A had an intermediate KD value of 31.3 ± 1.3 nM. Then, we used smFRET to further dissect the differences. Figure 1D shows that the FRET trace of 16bp 12nt-1 in RecQ 523 oscillated more frequently than that in RecQ. Besides, the RecQ 523 association mainly led to an intermediate at E0.74, which may reflect an unstable binding state on DNA (Figure 1E). We also compared the dwell time ton when helicase remains bound to DNA and the time interval toff between two successive binding events of RecQ, RecQ 523 , and the mixture of RecQ 523 and free HRDC. Both ton and toff followed the single-exponential decay with the average time ton′ and toff′ (Figure S1C-E). Although toff′ is almost the same between these three proteins, ton′ of RecQ is much longer, suggesting that RecQ binds to the DNA substrate more tightly than RecQ 523 . The equilibrium dissociation constant KD can also be obtained based on kon (1/toff′) and koff (1/ton′) [START_REF] Markiewicz | Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I[END_REF]. Indeed, the wild-type RecQ had a much lower KD than RecQ 523 (Figure 1F-H), consistent with the results from fluorescence polarization measurement shown in Figure S3.

Taken together, the above findings suggest that RecQ 523 associates with DNA in an unstable manner, therefore dissociates from the DNA more easily. HRDC can significantly reinforce RecQ binding on DNA by interacting with RecA core and ssDNA overhangs. Indeed, adding excess free HRDC to RecQ 523 restored the binding activity to some extent (Figures S1E,S3A, and Figure 1H), confirming the positive effect of the interaction between HRDC and the helicase core in DNA binding.

RecQ repetitively unwinds the 3′-partial duplex and fork DNA with moderate processivity.

After examining the DNA-binding activity, we characterized the duplex DNA unwinding mechanism of RecQ, as well as the influences of HRDC. Initially, 16bp 12nt-1 was used (Figure 2A, left panel), and upon addition of 5 nM RecQ and 20 μM ATP, two different types of traces were observed. First, the FRET value dropped from ~E0.9 to ~E0.5, representing the binding of RecQ to DNA; after an ~2.5 s dwell time, the signals of Cy3 and Cy5 disappear almost simultaneously, reflecting the one-step separation of the 16-bp duplex (Figure 2A, Figure S4A). Second, the FRET level oscillates until the signals of Cy3 and Cy5 disappear, reflecting complete duplex unwinding (Figure 2B). The initiation time between RecQ binding and duplex unwinding is referred to as t1 and has an average value of 2.4 s in 20 μM ATP (Figure 2C). The existence of t1 suggests that, instead of separating the duplex immediately upon arriving at the junction, RecQ may require several seconds to switch into the active unwinding state.

To observe the separation of two DNA strands in the duplex more directly, another substrate 16bp 12nt-2 was designed, in which both the donor and acceptor were labeled near the ss/dsDNA junction (Figure 2D, left panel). Figure S4B confirmed that the fluorophore on the translocation strand has little effect on RecQ. Upon addition of 5 nM RecQ and 20 μM ATP, both one-step unwinding (type I) and repetitive unwinding (type II) were observed (Figure 2D), consistent with the phenomena associated with 16bp 12nt-1. We used t2 to represent the time taken in the repetitive unwinding. With increases in ATP concentration, the fractions of one-step unwinding were slightly increased (Figure 2F). The co-existence of the two types may be attributed to the duplex length of 16bp 12nt-1 and 16bp 12nt-2 being close to the unwinding limit of RecQ. Therefore, in some cases, RecQ can unwind the duplex in one-step abruptly; while in other cases, RecQ may reach the limit and reverse the direction. To verify our speculation, another 3′-partial duplex with a 29-bp stem was designed (Figure 2G). In 5 nM RecQ and 20 μM ATP, continuous FRET fluctuations were observed in most cases, reflecting the repetitive unwinding by RecQ. With the increases in ATP concentration (Figure S4C), the unwinding fractions were significantly increased. Nevertheless, most traces still displayed the repetitive unwinding by RecQ before the complete unwinding (Figure S4D), reflecting the moderate processivity of RecQ even at 2 mM ATP. In addition, we discovered that fork DNA was also repetitively unwound by RecQ (Figure S5A). The processivity was over 20 bp in most cases, and RecQ may only occasionally arrive at the position beyond 28 bp (Figure S5B-E).

Figure 2H demonstrates the proposed mechanism of RecQ-catalyzed duplex unwinding. First, RecQ associates with the 3′-partial duplex or fork DNA at the 3′-ssDNA. Driven by ATP, RecQ translocates to the ss/dsDNA junction. Then, after a short initiation time, RecQ starts to unwind the duplex at a rapid speed. Once RecQ reaches the limit, it may loosen the tracking strand and switch to the 5′-ssDNA [START_REF] Bagchi | Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB[END_REF], translocate or slide back with the reannealing of the two strands, and then repetitively unwind the duplex.

HRDC domain suppresses the duplex DNA unwinding activity of RecQ.

To address the influence of HRDC on the duplex unwinding activity of RecQ, we directly measured the unwinding fractions of 16bp 12nt-1 and 16bp 12nt-2 by counting the number of Cy5 spots over time, as previously described [START_REF] Zhang | G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction[END_REF]. The remaining fractions with time in Figure 3A and Figure S6A both reflect that RecQ 523 displays a higher efficiency than RecQ in unwinding the 16-bp duplex.

We next analyzed the FRET traces of 16bp 12nt-1 and 16bp 12nt-2 in the three types of RecQ. Under our experimental conditions, HRDC has a negative impact on RecQ unwinding initiation (Figure 3B). For instance, the initiation time at 20 μM ATP is much longer for RecQ than for RecQ 523 (2.40±0.13 s versus 0.39±0.05 s, Figure 2C, and Figure S6B). In addition, the fractions of one-step unwinding in 16bp 12nt-2 by RecQ 523 are much higher than by RecQ (Figure 3C), i.e. the number of traces showing repetitive unwinding was greatly reduced without HRDC. The duration of repetitive unwinding in 16bp 12nt-2 by RecQ 523 was also significantly reduced compared with that by RecQ (1.13 s±0.03 versus 4.35±0.22 s, Figure S6C and Figure 2E).

Taken together, the above evidence indicates that the HRDC domain suppresses RecQ unwinding activity on duplex DNA mainly by increasing the unwinding initiation time and promoting repetitive unwinding. Importantly, RecQ Y555A , which abolishes the ssDNA-binding ability of HRDC, displays an activity level between that of the wild-type RecQ and RecQ 523 (Figure 3). Therefore, we speculate that the interactions of HRDC with the helicase core and with the ssDNA overhang both contribute to the weakened unwinding activity of RecQ by suppressing the ATP hydrolysis rate [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF] and promoting RecQ switching to the displaced strand (as indicated by a comparison of the fractions from the one-step unwinding by RecQ and RecQ Y555A ).

RecQ periodically patrols on the 5′-ssDNA overhang in 5′-partial duplex.

After systematically characterizing the unwinding of 3′-partial duplex and fork DNA by RecQ, we further examined the activity of RecQ on the 5′-partial duplex with the 47nt 17bp substrate (Figure 4A), in which Cy3 and Cy5 were labeled at the end of the 5′-overhang and the junction. Our previous studies have detected that the RecQ family helicases BLM [START_REF] Wu | BLM unfolds G-quadruplexes in different structural environments through different mechanisms[END_REF] and WRN [START_REF] Wu | Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding[END_REF], both possess the activity of anchoring at the 3′ ss/dsDNA junction and reeling in ssDNA by its 3′-5′ translocation activity with the 47nt 17bp substrate. DNA alone showed a stable EFRET at ~0.5 (Figure 4B, upper panel; Figure 4C). Upon addition of 5 nM RecQ and 20 μM ATP, the FRET value first dropped to ~0.2, reflecting the stretching of the ssDNA by RecQ, and then it occasionally increased to the values greater than 0.75 reflecting the looping of the 5′-ssDNA. At 2 mM ATP, the FRET bursts occurred more frequently (Figure 4B, lower panel). Figure S7 further suggests that the repetitive FRET fluctuations should be caused by the same helicase as excess protein was removed in the chamber. The FRET distributions of 47nt 17bp in 5 nM RecQ and 20 μM-2 mM ATP are shown in Figure 4C. Compared with DNA substrate alone, a new population at E0.21 emerged, consistent with the FRET decrease caused by RecQ binding in Figure 4B. There were also additional populations at higher FRET values, which were likely caused by the transient looping of the ssDNA.

Based on the above observations, we hypothesized that RecQ may anchor at the ss/dsDNA junction while translocating on the 5′-overhang in the 3′-5′ direction, thereby extruding an ssDNA loop. Upon arrival at the end of the 5′-ssDNA, RecQ may release the strand and restart a new cycle of translocation (Figure 4D). Besides, there is a 3-5 s interval between each FRET burst at 2 mM ATP (indicated by the green arrow in Figure 4B), suggesting that, after releasing ssDNA, RecQ requires a short time to restart a new cycle of ssDNA scanning.

HRDC domain significantly suppresses the periodical patrolling of RecQ on 5′-partial duplex.

Next, we examined the influence of HRDC on the periodical patrolling activity of RecQ on the 5′-partial duplex. In 5 nM RecQ 523 and 20 μM or 2 mM ATP, the FRET traces show similar bursts as that induced by RecQ, however with a much higher frequency (Figure 4E). The FRET values may increase to different levels possibly because of the release of ssDNA by RecQ before it reaches the 5′-end; however, most of the bursts were greater than E0.75 (Figure 4B,4E). Therefore, to better quantify the fractions of DNA in the looping state as shown in Figure 4F, an artificial threshold was set at E0.75, above which loops were presumed to be extruded. Figure 4G shows that with increases in ATP concentration, the fractions of DNA with EFRET values above 0.75 increased significantly. More importantly, they were highest in RecQ 523 , and lowest in RecQ at each ATP concentration (Figure 4G), reflecting the much stronger periodical patrolling activity of RecQ 523 . The number of FRET bursts in the 20-s time window was also quantified (Figure 4H). The frequency of looping events increased significantly with the increases in ATP concentration. Moreover, the frequencies were much higher in RecQ 523 than in RecQ, highlighting the extraordinary patrolling activity of RecQ 523 .

By comparing the FRET traces in Figure 4B, 4E, we noticed that the time intervals between individual FRET bursts were much decreased in RecQ 523 than in RecQ at both low or high ATP concentrations. This evidence indicates that HRDC significantly prolongs the time for RecQ to restart the next cycle of translocation, consistent with the negative impact of HRDC on the initiation of duplex DNA unwinding, as shown in Figure 3B. As both the initiation time in duplex unwinding and time interval in periodical patrolling depend on ATP concentration, the existence and duration of these times may be related to ATP hydrolysis, as HRDC suppresses ATP hydrolysis by interacting with the RecA core [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF], leading to a longer unwinding initiation and patrolling restart time. RecQ Y555A displays a medium level activity between RecQ and RecQ 523 (Figure 4F-H), suggesting that the interaction of HRDC with the 5′-ssDNA also contributes to the decrease in the periodical patrolling activity of RecQ. It is possible that HRDC dynamically binds onto the 5′-ssDNA ahead of RecQ, inhibiting RecQ translocation on the strand to some extent.

RecQ unfolds G4 structure in a stepwise and repetitive fashion and maintains G4 DNA in an unfolded state for a long time.

As the E. coli genome indeed includes considerable amounts of G4s which may play important roles in critical cellular processes [START_REF] Kaplan | G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch[END_REF][START_REF] Teng | Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication[END_REF], here we further investigated RecQ-catalyzed G4 unfolding with or without the HRDC domain. Unexpectedly, RecQ displayed a very poor affinity towards the G4 structure with the KD value of 658.2±79 nM (Figure S8A). Figure S8A further shows that a 3′-ssDNA overhang is indispensable for RecQ to efficiently associate with the G4 DNA. Therefore, we speculated that RecA1 and RecA2 domains in RecQ may first bind to the 3′-ssDNA region (~10 nt), anchoring the helicase onto the substrate, then RQC can interact with the G4 structure, consistent with the structure of C. sakazakii RecQ in complex with G4 DNA [START_REF] Voter | A guanine-flipping and sequestration mechanism for G-quadruplex unwinding by RecQ helicases[END_REF].

Next, we carried out smFRET unwinding assay with the substrate 29bpG4 12nt in which the G4 motif was linked with a 29-bp duplex at its 5′ end and a 12-nt ssDNA at its 3′ end (Figure 5A) as previously reported [START_REF] Wu | BLM unfolds G-quadruplexes in different structural environments through different mechanisms[END_REF]. Cy3 was labeled at the nucleotide between the G4 motif and 3′ tail, and Cy5 was labeled 6 bp inside the duplex. The fluorophores were so spaced that the FRET signal can sensitively report the conformational change of G4 [START_REF] Wu | BLM unfolds G-quadruplexes in different structural environments through different mechanisms[END_REF]. In 100 mM KCl, the FRET value of 29bpG4 12nt remained at a stable level at ~0.9 (Figure S8B), reflecting the well folding of the G4 structure. Therefore, this buffer condition was used for further RecQ-catalyzed unfolding experiments.

The fractions of remaining DNA molecules versus the time after the addition of 5 nM RecQ and different concentrations of ATP were determined. Figure 5B indicates that RecQ should be able to unfold the G4 structure in the presence of ATP; otherwise, the downstream duplex cannot be unwound (Figure 5B). Then, the FRET traces of 29bpG4 12nt after the addition of different concentrations of RecQ and ATP were recorded and analyzed (Figure 5C). In the absence of ATP, no change was detected in the FRET distributions even at 100 nM RecQ. However, at 5 nM RecQ and 20 μM ATP, the population at ~E0.9 decreased significantly, accompanied by an increase in low-range FRET populations, reflecting the disruption of the G4 structure. Both pieces of evidence indicate that RecQ-catalyzed G4 unfolding was ATP-dependent. Figure 5D shows that the FRET traces in 5 nM RecQ and 20 μM ATP switch between at least four states, suggesting the disruption and dynamic conversion of G4 structure between different folding states (the duration time was defined as ton*). Then, the FRET value returns to the original level, likely because of the dissociation of RecQ. After a short interval, toff*, another cycle of similar FRET fluctuation begins. The distributions of both ton* and toff* follow the single-exponential decay with an average time of ~8 s (Figure 5E). To further understand the different unfolding states, we selected the fluctuation regions within ton* from ~200 traces and plotted the FRET histograms (Figure 5F). Three peaks can be discriminated: the leftmost peak at E0.33 could be the ssDNA [START_REF] Wu | BLM unfolds G-quadruplexes in different structural environments through different mechanisms[END_REF], while the other two peaks, at E0.52 and E0.65, may represent the proposed G-hairpin and G-triplex structures, respectively [START_REF] Wu | BLM unfolds G-quadruplexes in different structural environments through different mechanisms[END_REF][START_REF] Hou | Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase[END_REF][START_REF] Zhou | Involvement of G-triplex and G-hairpin in the multi-pathway folding of human telomeric G-quadruplex[END_REF](42). The transition density plot in Figure 5G further indicates that the transitions between the above states are reversible; i.e., the completely or partially unfolded G4 can refold back to a more complete state while RecQ remains associated with the G4 motif. Figure S9 further confirms the G4 unfolding activity of RecQ with a substrate in which the G4 motif was at the 5′ end of the partial duplex.

A reasonable interpretation of the observed FRET oscillation is presented in Figure 5H. RecQ unfolds the G4 structure into ssDNA in a stepwise manner with at least two intermediate states, G-triplex and G-hairpin. It is worth noting that this is a simplified model, highlighting that there are multiple states in the dynamic interaction between RecQ and G4; however, the specific structures of those states still need further ascertainment by other methods. Besides, our results further show that RecQ can maintain the G4 structures in unfolded states for a relatively long time (~8 s at 20 μM ATP), and it may be alike to that observed from FANCJ helicase, which can recognize G-quadruplexes and mediate their longstanding stepwise unfolding in repeating cycles [START_REF] Wu | G-quadruplex recognition and remodeling by the FANCJ helicase[END_REF]. However, according to previous reports, the G4 structure was transiently unfolded by Pif1 (~1 s) [START_REF] Hou | Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase[END_REF] and DHX36, BLM, WRN (~2 s) [START_REF] Tippana | Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases[END_REF][START_REF] Chen | Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36[END_REF] during the quick and frequent switching between well-folded and unfolded states.

The HRDC domain is essential for the complete and long-time unfolding of the G4 DNA structure by RecQ. We next examined the influence of HRDC on the G4-unfolding activity of RecQ. First, the fractions of 29bpG4 12nt on coverslip versus the time after the addition of 5 nM helicase and 2 mM ATP was determined (Figure 6A). Although RecQ displayed the lowest duplex unwinding activity among the three proteins (Figure 3A), the unwinding of 29bpG4 12nt by RecQ was more efficient than by RecQ 523 , suggesting that HRDC plays a positive role in G4 unfolding.

Afterward, FRET traces of 29bpG4 12nt after the addition of 5 nM RecQ and 2 mM ATP were analyzed. In all three types of RecQs, the FRET distributions of 29bpG4 12nt showed shifts to the lower band (Figure 6B), indicating the unfolding of G4. To quantify the differences between them, an artificial threshold at E0.65 corresponding to the G-triplex state in Figure 5F was set, below which G4 structure is considered as partially or completely unfolded. Figure S8C shows that the unfolding fractions by RecQ are much higher than by RecQ 523 , highlighting the importance of HRDC in G4 unfolding. The FRET traces in 5 nM RecQ 523 and 20 μM ATP were consistent with that in RecQ, with the FRET value switching between different states (Figure 6C). Then, we selected the regions showing oscillations and constructed FRET histograms (Figure 6D). The P1 state corresponding to ssDNA is the lowest in RecQ 523 (Figure 6E, 14% versus 52% in RecQ). Instead, in RecQ 523 , most of the molecules are at P2 (55%) and P3 (41%) state which may correspond to G-hairpin and G-triplex; i.e., RecQ 523 most likely disrupts G4 into partially unfolded states and can barely disrupt G4 completely.

We also compared the unwinding time ton* and the time interval toff* of FRET traces in the three types of RecQs (Figure 6F-G). Under the same experimental conditions, ton* in RecQ was at least two-fold longer than that in RecQ 523 , indicating that RecQ 523 was more prone to dissociate from the G4 substrate after partially disrupting G4. On the other hand, toff* in RecQ was shorter than that in RecQ 523 ; i.e., RecQ 523 takes a longer time to re-associate with the G4 substrate and restart the unfolding.

The differences between RecQ and RecQ 523 reflect that the HRDC domain substantially promotes G4 unfolding by increasing the duration time of each unfolding event in parallel with increasing the degree of G4 disruption. Unexpectedly, the G4 unfolding activity of RecQ Y555A is very similar to that of the wild-type RecQ (Figure 6), implying that the interaction between HRDC and ssDNA may have very little impact. To further dissect whether HRDC directly interacts with the G4 structure and whether RecQ 523 binds G4 differently than RecQ, we measured the binding affinity between those proteins and the G4 structure. Figure 6H-I indicates that, although HRDC itself cannot interact with the G4 structure, both RecQ and RecQ Y555A bind to the G4 substrate with 3′-ssDNA much stronger than RecQ 523 . Therefore, the reinforcement of the association of RecQ on the G4 substrate may be mainly caused by the interaction between HRDC and helicase core.

DISCUSSION

Proposed roles of the HRDC domain in regulating the helicase activity E. coli RecQ on different nucleic acid substrates.

The functions of HRDC in duplex DNA unwinding have been studied previously.

Harami et al. reported that HRDC in E. coli

RecQ suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF]. Using magnetic tweezers, the same group then discovered that HRDC mediates pausing and shuttling during hairpin DNA unwinding [START_REF] Harami | Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination[END_REF]. Later, Bagchi et al. reported that RecQ unwinds hairpin DNA using a fast mode of continuous unwinding and a slow mode of persistent random walking, and the deletion of HRDC diminished the slow mode [START_REF] Bagchi | Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB[END_REF]. In our current smFRET study without external forces, RecQ mainly displays repetitive unwinding/rewinding activity on 3′-tailed duplex and fork DNA; therefore, we focused on the influence of HRDC on the repetitive unwinding behavior of RecQ. More importantly, we also carried out an in-depth analysis of the impact of HRDC on the repetitive ssDNA patrolling and G4 unfolding activities of RecQ.

We suggest that HRDC suppresses the duplex DNA unwinding activity of RecQ with the proposed role as shown in Figure 7A. First, HRDC increases the initiation time of RecQ for duplex unwinding (Figure 3B). The increase in initiation time with the existence of HRDC is possibly due to that HRDC dynamically interacts with the RecA core, thereby inhibiting ATP hydrolysis [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF]. As the HRDC binding site is near the ATP binding cleft of RecA domains, it is also possible that ATP binding or ADP and/or Pi release is inhibited [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF]. Second, more frequent repetitive unwinding was observed with RecQ while more unidirectional unwinding was observed with RecQ 523 or RecQ Y555A (Figure 3C). This is attributed to HRDC's dynamic interaction with the 5′-ssDNA, which promotes the strand-switching activity of RecQ.

HRDC also significantly suppresses the repetitive ssDNA patrolling activity of RecQ on a 5′-tailed duplex. The major difference between RecQ and RecQ 523 is in the patrolling frequency and burst width (Figure 4), which are related to ATP concentration, suggesting that HRDC may slow down the switch of RecQ into an active translocation state as well as the translocation rate by inhibiting ATP binding, ATP hydrolysis, or ADP and/or Pi release [START_REF] Harami | The HRDC domain of E-coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling[END_REF], as mentioned above. It is also possible that the dynamic binding of HRDC to the ssDNA ahead of RecQ inhibits its translocation initiation, thus partially contributing to the reduced patrolling frequency (Figure 7B).

In sharp contrast with the inhibiting effect of HRDC on duplex unwinding and repetitive ssDNA patrolling, our results demonstrate that HRDC is essential for the complete and long-time unfolding of G-quadruplex DNA by RecQ. Considering that the G4 unfolding activity of RecQ Y555A is very similar to that of RecQ but more efficient than RecQ 523 (Figure 6), the positive impact of HRDC during G4 unfolding should be mainly caused by the interaction of HRDC with the RecA core. Therefore, we speculate that HRDC is crucial for RecQ to proceed with G4 unfolding by reinforcing the association of RecQ with the DNA substrate through interacting with RecA core, thus ensuring the complete unfolding of the G4 structure (Figure 7C).

It is worth noting that, the HRDC domain in N. gonorrhoeae RecQ has been previously reported to improve G4 unfolding [START_REF] Cahoon | Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation[END_REF]; however, this RecQ helicase has three tandem HRDCs, leading to very complex and different functions compared with other RecQ helicases. Indeed, both N. gonorrhoeae RecQ and its version with two HRDCs deleted bind to G4 relatively well with KD = 55.1 and 86.5 nM [START_REF] Cahoon | Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation[END_REF], respectively; however, E. coli RecQ binds to G4 very poorly with KD = 658.2 nM (RecQ 523 can barely bind to G4), and a 3′-ssDNA is required for the RecA domains to bind first, anchoring the helicase onto the substrate. Moreover, N. gonorrhoeae RecQ takes ~211 s to unfold 50% of the G4 structures, reflecting a very poor G4 unfolding activity [START_REF] Cahoon | Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation[END_REF]. Altogether, there are essential differences between these two helicases, and we think that E. coli RecQ is an ideal helicase to address the in-depth functional mechanism of HRDC as most RecQ family members only have one HRDC.

HRDC may play a role in mediating the cooperative binding of RecQ onto DNA substrates.

Different RecQ family members may exhibit different oligomeric state in solution. As for E. coli RecQ, our previous studies [START_REF] Xu | The Escherichia coli RecQ helicase functions as a monomer[END_REF][START_REF] Zhang | Escherichia coli RecQ is a rapid, efficient, and monomeric helicase[END_REF] found that it is monomeric in solution up to a concentration of 20 μM, this property is not affected by the presence of ATP. Although RecQ unwinds DNA as a monomer, our further study [START_REF] Li | Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: a fluorescence cross-correlation spectroscopy study[END_REF] and Kowalczykowski group's research [START_REF] Harami | Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination[END_REF] both found that multiple E. coli RecQ monomers can cooperate to unwind long DNA substrates, dependent on the protein concentration. In this study, we mainly used 5 nM protein concentration to detect the binding initiation, dsDNA unwinding, 5' -ssDNA translocation, and G4 unwinding process; therefore, we treated RecQ as a monomer, similarly to the previous single-molecule studies [START_REF] Harami | Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination[END_REF][START_REF] Bagchi | Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB[END_REF].

We have noticed that the HRDC domain not only significantly enhances the binding affinity between RecQ and DNA substrate by interacting with the RecA core and ssDNA overhangs, but also increases the cooperativity between different RecQ molecules in DNA binding. As shown in Figure S3, the Hill coefficient is the lowest for RecQ 523 among the three helicases (RecQ, RecQ 523, and RecQ Y555A ). Meanwhile, adding free HRDC to RecQ 523 has little effect on the Hill coefficient (Figure S3). Therefore, we speculate that, due to the long flexible linker, HRDC might also be able to dynamically interact with the RecA core of another RecQ nearby, leading to the relatively high cooperativity of RecQ molecules in DNA binding. It is also possible that multiple RecQ monomers may unwind the duplex DNA and G4 DNA with the cooperative translocation at high protein concentration.

Potential biological significances of HRDC in modulating RecQ activities in DNA repair.

Our results indicate that HRDC suppresses the dsDNA unwinding activity of RecQ although it reinforces RecQ binding to DNA. Besides, HRDC significantly promotes the repetitive unwinding of the duplex by RecQ with a moderate processivity. As E. coli RecQ is a central DNA recombination and repair helicase, the above observations suggest that HRDC may play a positive role in improving the precision and efficiency when RecQ removes the short duplex invasions in D-loops formed in the illegitimate homologous recombination [START_REF] Hanada | RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli[END_REF]. What's more, as HRDC can significantly strengthen RecQ binding to the G4 substrate, and ensure complete and long-time unfolding of G4 structure, we speculate that HRDC may be crucial for some key biological processes mediated by RecQ such as repairing stalled replication forks induced by G4 [START_REF] Kaplan | G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch[END_REF][START_REF] Teng | Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication[END_REF]. In brief, our study reveals that the auxiliary structural component HRDC differentially modulates the activities of E. coli RecQ in processing different DNA structures by dynamically interacting with the RecA core and ssDNA, and provides new insights into the functioning of RecQ during DNA replication, recombination, and repair.

EXPERIMENTAL PROCEDURES

Buffers.

The RecQ reaction buffer contained 50 mM KCl, 2 mM MgCl2, 1 mM DTT and 0.1 mg/mL BSA in 20 mM Tris-HCl (pH 7.5), unless otherwise specified. For single-molecule measurements, 0.8% D-glucose, 1 mg/ml glucose oxidase (266600 units/g, Sigma-Aldrich, St Louis, MO, USA), 0.4 mg/ml catalase (2000-5000 units/mg, Sigma-Aldrich) and 1 mM Trolox (Sigma-Aldrich) were added to the reaction buffer.

DNA constructs.

All oligonucleotides required to generate DNA substrates were purchased from Sangon Biotech (Shanghai, China). The sequences and labeling positions of all the oligonucleotides are listed in Table S1. For DNA constructs used in single-molecule measurements, DNA was annealed with a 1:3 mixture of stem and ssDNA or G4 strands by incubating the mixture at 95°C for 5 min, then slowly cooling down to room temperature in about 7 hours. The strand without biotin was used in excess to reduce the possibility of having non-annealed strands anchored to the coverslip surface. The concentration of the stem strand was 2.5 μM and all annealing was carried out in the annealing buffer containing 100 mM KCl and 20 mM Tris-HCl (pH 7.5). The partial duplex DNA used in the equilibrium DNA binding assay was annealed with a 1:1 mixture of the two strands.

Protein expression, purification, and labeling.

The expression and purification of E. coli RecQ, RecQ 523 , and RecQ Y555A were carried out as described previously [START_REF] Kocsis | A nucleotide-dependent and HRDC domain-dependent structural transition in DNA-bound RecQ helicase[END_REF]. For simplicity, we refer to E. coli RecQ as RecQ herein. In brief, each RecQ construct was cloned into the pET15b-SUMO vector and expressed in BL21 (DE3) induced by 0.3 mM IPTG at 18℃ for 16 h, respectively. Then, the recombinant protein was severally purified by Ni affinity chromatography; after digested by SUMO protease at 4°C overnight, each RecQ construct was purified again by Ni affinity chromatography to remove 6*His and SUMO tag. The protein purity was more than 95% determined by SDS-PAGE as described previously [START_REF] Kocsis | A nucleotide-dependent and HRDC domain-dependent structural transition in DNA-bound RecQ helicase[END_REF]. The protein concentration was more than 5 mg/ml determined by UV280 using Thermo Scientific Nanodrop 2000c. When measuring the protein concentration, the Molar extinction coefficients of RecQ, RecQ Y555A , RecQ 516 , HRDC were 48820 M -1 cm -1 , 47330 M -1 cm -1 , 44560 M -1 cm -1 , 2560 M -1 cm -1 , respectively.

As RecQ contains 11 cysteine residues, it is difficult to specifically label the HRDC domain with a single fluorophore. To avoid nonspecific labeling, we depended on the flexible linker (~22 aa) between the RQC and HRDC domains to establish a scheme based on sortase A ligation [START_REF] Mao | Sortase-mediated protein ligation: a new method for protein engineering[END_REF], as our previous report [START_REF] Teng | A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay[END_REF]. In brief, recombinant protein NH2-GGG-HRDC E610C labeled with a single Cy5 fluorophore (Lumiprobe Inc., Baltimore, MD, USA) and RecQ 1-516 -LPETG were separately prepared and then ligated by sortase A in ligation buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl and 20 mM CaCl2) at 34°C for 1 hour. The final ligated protein was purified and stored at 80°C.

Single-molecule fluorescence data acquisition.

The smFRET assay was performed as described previously [START_REF] Wu | Single-molecule technique: a revolutionary approach to exploring fundamental questions in plant science[END_REF]. 5 nM protein concentration was mainly used in our experiment unless otherwise specified. Imaging was initiated before RecQ and ATP were flowed into the chamber. We used an exposure time of 100 ms for all measurements at a constant temperature of 22°C. To determine the fractions of unwound DNA with time, a series of 1-s movies were recorded at different times, and the Cy5 spots were counted to represent the number of remaining DNA molecules.

FRET data analysis.

The FRET efficiency was calculated using E = IA/(ID+IA), where ID and IA represent the intensities of the donor and acceptor, respectively. Basic data analysis including transition density plot (TDP) was carried out by scripts written in MATLAB. All data fitting was conducted with Origin 8.0. An automated step-finding method (from http://bio.physics.illinois.edu/HaMMy.asp) was used to characterize RecQ's association with and dissociation from partial duplex DNAs as well as the stepwise patterns observed in G4 unfolding. The histograms of FRET efficiency and dwell time from more than 300 molecules were fitted with multi-peak Gaussian distribution and single-exponential decay, respectively.

Equilibrium DNA binding assay.

Binding of RecQ to DNA was analyzed by fluorescence polarization assay using Infinite F200 PRO (TECAN, Zürich, Switzerland) at a constant temperature of 25°C [START_REF] Dou | The DNA binding properties of the Escherichia coli RecQ helicase[END_REF]. DNA labeled with FAM was used in this study (Table S1). Varying amounts of protein were added to a 150-μl aliquot of binding buffer containing 5 nM DNA. Each sample was allowed to equilibrate for 5 min, and the fluorescence polarization value was then measured. The binding curve was fitted by the Hill equation: y = [RecQ] n /(KD n + [RecQ] n ), where y is the binding fraction, n is the Hill coefficient, and KD is the apparent dissociation constant.

FIGURE CAPTIONS

9 K 1 [Figure 1 .

 911 Figure 1. RecQ helicase repetitively associates with and dissociates from the 3′-partial duplex DNA. (A) Crystal structures of an HRDC-deleted E. coli RecQ (PDBid 1OYW), HRDC-deleted C. sakazakii RecQ in complex with DNA (4TMU), and HRDC-containing human BLM in complex with DNA (4O3M). (B) Domain map of E. coli RecQ constructs used in this study. (C) Schematic set-up of the smFRET experiment. (D) The typical smFRET traces of 16bp 12nt-1 in 50 nM RecQ and RecQ 523 . An automated step-finding algorithm was used to identify the different FRET states (red line). (E) FRET histograms of 16bp 12nt-1 in 50 nM RecQ or RecQ 523 based on the fitted FRET traces. In all following figures, the FRET histograms were collected from more than 200 traces.(F-H) The dissociation rate (koff =1/ton′, red) and the binding rate (kon=1/toff′, black) as a function of protein concentrations. As expected for a binary reaction, the dissociation rate is independent of protein concentration while the binding rate has a linear dependence on it. The dissociation constant is thus determined as KD = koff /kon. Error bars denote the standard deviations.

Figure 2 .

 2 Figure 2. Unwinding of duplex DNA by RecQ. (A-B) The typical fluorescence and FRET traces of 16bp 12nt-1 in 5 nM RecQ and 20 μM ATP. More reaction traces were exhibited in Figure S4A. t1 represented the initiation time before duplex unwinding. (C) Distribution of t1 obtained from single exponential fitting. all the following figures, the time histograms were collected from more than 200 traces. (D) Two typical FRET traces of 16bp 12nt-2. Type I represents one-step unwinding, and type II represents repetitive unwinding with total unwinding time t2. (E) Distribution of t2 obtained from single exponential fitting. (F) The fractions of type I increase with the increase in ATP concentrations. (G) Repetitive unwinding can be observed in the substrate 29bp 26nt. (H) A proposed model for RecQ-catalyzed duplex unwinding.

Figure 4 .Figure 5 .Figure 7 .

 457 Figure 4. RecQ periodically patrols on the 5′-ssDNA. (A) The 5′-partial duplex 47nt 17bp contains a 17 bp duplex and a 47 nt 5′-ssDNA. (B) The typical FRET traces of 47nt 17bp in 5 nM RecQ and indicated ATP concentrations. (C) FRET distributions of 47nt 17bp only, and in 5 nM RecQ and different concentrations of ATP. (D) A proposed action of RecQ on the 5′partial duplex. (E) The typical FRET traces of 47nt 17bp in 5 nM RecQ 523 and indicated ATP concentrations. (F) FRET distributions of 47nt 17bp in 5 nM RecQ, RecQ 523, and RecQ Y555A with 2 mM ATP. (G) The fractions of DNA at looping states (FRET>0.75) in different reaction conditions. (H) The number of FRET bursts within the 20 s time window in 5 nM RecQ, RecQ 523 , RecQ Y555A with indicated ATP concentration.

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ACKNOWLEDGEMENTS

We thank Dr. Wen-Qiang Wu at Henan University for insightful discussions.

DATA AVAILABILITY

All data are contained within the manuscript.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.