Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection

Chaoyou Xuea, Weibin Wangb,c, J. Brooks Crickarda, Corentin J. Moevusd, Youngho Kwonb,c, Patrick Sungb,c, and Eric C. Greenea,1

aDepartment of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032; bDepartment of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520; cDepartment of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; and dDepartment of Pathology and Cell Biology, Columbia University, New York, NY 10032

Edited by James E. Haber, Brandeis University, Waltham, MA, and approved February 6, 2019 (received for review November 9, 2018)

In the repair of DNA double-strand breaks by homologous recombi- Eukaryotic DNA end resection involves three nucleases nation, the DNA break ends must first be processed into 3′ single- (Mre11, Exo1, or Dna2) and a RecQ-related DNA strand DNA overhangs. In budding yeast, end processing requires (Sgs1 in budding yeast, and either BLM or WRN in humans) the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, (21–24). During the initial stages of end resection, Sae2 (CtIP in Top3-Rmi1, and (RPA). Here, we use single- humans) activates the Mre11–Rad50–Xrs2 (Mre11–Rad50– ′ molecule imaging to visualize Sgs1-dependent end processing in Nbs1 in mammals) complex to endonucleolytically cleave the 5 ′ → ′ real-time. We show that Sgs1 is recruited to DNA ends through strand close to the DSB, followed by limited 3 5 exonu- – – cleolytic action to create a short DNA gap that serves at the Top3-Rmi1 dependent or independent means, and in both cases – Sgs1 is maintained in an immoble state at the DNA ends. Impor- entry site for the long-range resection machinery (17, 25 28). Long-range resection is mediated via alternate pathways that engage tantly, the addition of Dna2 triggers processive Sgs1 translocation, ′ → ′ but DNA resection only occurs when RPA is also present. We also either Exo1 or Dna2 (12, 17, 19, 28). Exo1 is a 5 3 exonuclease (12, 17–19). While Dna2 possesses 5′ and 3′ endonuclease activities demonstrate that the Sgs1–Dna2–Top3-Rmi1–RPA ensemble can effi- and has a modest DNA helicase activity, only the nuclease function ciently disrupt nucleosomes, and that Sgs1 itself possesses nucleo- is required for end resection in otherwise wild-type cells. It has been some remodeling activity. Together, these results shed light on the shown that Dna2 incises ssDNA generated via DNA unwinding by regulatory interplay among conserved protein factors that mediate the Sgs1 helicase (12, 19, 21, 22). Sgs1 is a member of the RecQ BIOCHEMISTRY the nucleolytic processing of DNA ends in preparation for homolo- helicase family and is orthologous to the human BLM helicase gous recombination-mediated chromosome damage repair. (29–31), which also cooperates with DNA2 in DNA end resection in human cells (24). There is emerging evidence that WRN, another DNA repair | | single molecule | helicase | RecQ family helicase, functions in parallel to BLM in DNA DNA end resection end resection (17, 19, 32–34). Mutations in BLM and WRN lead to Bloom and Werner syndromes, respectively, which are associ- NA double-strand breaks (DSBs) are highly toxic DNA le- ated with severe developmental defects, elevated levels of Dsions, which have the potential to cause the types of chro- chromosomal rearrangements, and early-onset cancers. Patients mosomal rearrangements found in cancer cells (1–3). DSBs are of Werner syndrome also display marked premature aging (14, induced by ionizing radiation and drugs used in cancer therapeutics, 30, 31). The chromosomal abnormalities observed in cells from and also as a consequence of replication fork stalling or collapse and BLM patients are strikingly similar to those in sgs1 mutant cells, aptly highlighting the conserved function of these in transcription–replication conflicts stemming from the formation of – genome maintenance (29). R-loops (3 6). In contrast, programmed meiotic DSBs introduced by Regulatory factors of Sgs1-dependent DSB processing have been the conserved Spo11 complex are crucial for establishing stable linkage described, but their mechanism of action remains poorly understood between homologous chromosomes to promote disjunction of the (12, 17, 19). Sgs1 associates with the type IA topoisomerase Top3 paired chromosomes in the first meiotic division (7–9). Homologous recombination (HR) encompasses interrelated but Significance mechanistically distinct pathways, namely, synthesis-dependent strand-annealing, break-induced replication, the DSB repair path- way that can yield chromosome arm cross-overs, and the double DNA end resection is key to determining whether a DNA dissolution pathway (3, 10–12).Theimportanceof double-stranded DNA break (DSB) will be repaired by a high- HR to genome integrity is evidenced by the prevalence of cancers, fidelity or error-prone pathway. Here, we establish a single- cancer-prone syndromes, and other severe genetic disorders in pa- molecule assay for directly observing the resection of DSBs by tients with mutations in HR proteins (13–16). conserved protein factors from the budding yeast Saccharo- HR is reliant upon the highly conserved RAD52 epistasis myces cerevisiae. Our work defines important parameters of group of proteins, including the DNA recombinase Rad51, an the end-resection process that precedes high-fidelity repair, ATP-dependent DNA-binding protein that assembles into long such as resection speed and distance, and also unveils mecha- helical filaments on single-stranded DNA (ssDNA) (11, 12). The nistic features of how the RecQ helicase Sgs1 is targeted to resulting Rad51-ssDNA nucleoprotein filament is referred to as DSBs and how Sgs1 is activated by regulatory proteins. the presynaptic complex and is capable of catalyzing homologous DNA pairing that yields heteroduplex DNA joints with donor Author contributions: C.X. and E.C.G. designed research; C.X. performed research; W.W., duplex molecules (12). The aforementioned HR pathways all J.B.C., C.J.M., and Y.K. contributed new reagents/analytic tools; C.X., P.S., and E.C.G. analyzed data; and C.X., W.W., Y.K., P.S., and E.C.G. wrote the paper. share an initial requirement for extensive 5′ → 3′ resection of DNA ends (12, 17–19). This 5′ strand resection step yields long 3′ The authors declare no conflict of interest. ssDNA tails for the activation of the Mec1/ATR DNA damage This article is a PNAS Direct Submission. checkpoint and serves as the template for assembling Rad51 Published under the PNAS license. presynaptic complexes (12, 17–19). Thus, DNA end resection rep- 1To whom correspondence should be addressed. Email: [email protected]. resents a crucial regulatory point for determining whether a DSB This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. will be repaired by HR that is mostly error-free, as opposed to more 1073/pnas.1819276116/-/DCSupplemental. error-prone nonhomologous DNA end-joining (17, 19, 20). Published online March 8, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1819276116 PNAS | March 26, 2019 | vol. 116 | no. 13 | 6091–6100 Downloaded by guest on September 29, 2021 (Topo IIIα in humans) and the oligonucleotide/oligosaccharide- AB binding (OB)-fold containing protein, Rmi1, to form the STR (Sgs1–Top3-Rmi1) complex (35). Top3 and Rmi1 are both required for optimal activity of Sgs1–Dna2 during DSB processing (22). The equivalent of the STR complex in humans, referred to as the BTR complex, harbors BLM, Topo IIIα,RMI1,andasecondOB-fold protein, RMI2 (33, 36–40). STR and BTR also participate in the dissolution of double Holliday junctions that can arise during a late C D stage of HR, and this function of STR/BTR requires the top- oisomerase activity of Top3/Topo IIIα (12, 19). In contrast, the topoisomerase activity of Top3/Topo IIIα is dispensable for DSB resection (22, 39). Replication protein A (RPA) is an ssDNA- binding protein that regulates Dna2 nuclease activity to enhance 5′ strand cleavage while attenuating degradation of the 3′ strand (22–24). MRX and Top3-Rmi1 both stimulate resection efficiency E FG by enhancing Sgs1 helicase activity (22, 23). MRX and Top3 also recognize DNA ends, perhaps enabling these factors to target Sgs1 to DSBs through protein–protein interactions (39, 41). Biochemical reconstitution of DNA end resection has expanded our molecular understanding of this process (22–24). However, there exist wide knowledge gaps regarding the mechanisms of end resection and we have only a rudimentary understanding of how protein–protein interactions help regulate DNA end resection. Here, we use real-time single-molecule imaging to examine the interplay among proteins involved in the Sgs1-dependent DSB HI J end-resection pathway. Most notably, we show that Sgs1 is selec- tively targeted to double-stranded DNA (dsDNA) ends through two distinct pathways: either in the ATP-free apo state or through a Top3-Rmi1–dependent mechanism when ATP is present. We show that the DNA end-bound Sgs1 remains in an inert confor- mation until the arrival of Dna2, which activates Sgs1 for rapid and highly processive translocation. Surprisingly, Dna2-mediated activation of end-bound Sgs1 yields two distinct populations of complexes, as evidenced by a bimodal distribution of translocation velocities. The addition of RPA not only triggers nucleolytic di- gestion of DNA by Dna2 and the accumulation of RPA at the resulting ssDNA overhangs, but it also gives rise to a single pop- ulation of end-processing complexes with a defined velocity dis- tribution. Finally, we show that the Sgs1-driven end-resection Fig. 1. Real-time visualization of Sgs1 activity on dsDNA. (A) Schematic of machinery exerts enough force to rapidly displace nucleosomes double-tethered DNA curtains assay with Sgs1. (B) Schematic of Sgs1 un- from its path of movement. These observations thus support a winding dsDNA as revealed by the binding of RPA-mCherry. (C) Kymograph model in which nucleosomes are pushed away from the resected showing Sgs1 (unlabeled) unwinding dsDNA (unlabeled) as revealed by the DNA ends, which has important implications for how the for- binding of RPA-mCherry (magenta); note that buffer flow was OFF during mation of DSBs may affect the retention of epigenetic histone data collection. Arrowheads indicate the initiation sites where Sgs1 initiates modifications. Taken together, our findings furnish insights into dsDNA unwinding. (D) Distribution of initiation sites for Sgs1 unwinding (n = the network of regulatory interactions that target the end-processing 100). (E) Quantification of Sgs1 translocation directionality. B→P indicates machinery to DSBs and help to constrain the activities of both movement in the direction from the barrier to the pedestal, and P→B rep- Sgs1 and Dna2 until the assembly of end-processing machinery resents movement in the opposite direction. (F) Velocity distribution of has been completed. Sgs1 with RPA-mCherry unwinding rates on double-tethered dsDNA. The solid blue line represents a Gaussian fit to the data. (G) Survival probability Results plot of Sgs1 translocation in the presence of RPA-mCherry on double- tethered dsDNA. (H) Kymograph showing an example of GFP-Sgs1 (green) DNA Binding and Unwinding Activities of Sgs1. We used DNA curtain unwinding dsDNA (unlabeled) in the presence of RPA-mCherry (magenta); assays and total internal reflection fluorescence (TIRF) microscopy note that buffer flow was OFF during data collection. The white arrowheads to visualize the behavior of Sgs1 on individual dsDNA molecules indicate the site of initiation. (I) Sgs1 velocities measured with different (Fig. 1A). For initial assays, we utilized double-tethered dsDNA combinations labeled and unlabeled Sgs1 and RPA, as indicated. Error bars curtains with λ-DNA (∼48.5 kb) as substrate to test for Sgs1 heli- represent SD obtained through Gaussian fits. (J) Sgs1 velocities measured case activity (Fig. 1A) (42). We employed RPA-mCherry to reveal with different combinations labeled and unlabeled proteins, as indicated. ssDNA strands emanating from dsDNA unwinding catalyzed by Error bars represent SD obtained through Gaussian fits. Sgs1 (unlabeled) (Fig. 1B). Coinjection of Sgs1 (0.2 nM) and RPA- mCherry (2 nM) led to the appearance of RPA-mCherry signal on the dsDNA molecules (Fig. 1C). RPA-mCherry appeared on the more) Sgs1 molecules bound in close proximity to one another, and dsDNA and then spread outward from these sites to yield dis- then translocated in opposite directions along the DNA. Analysis tinctive wedge-shaped patterns (Fig. 1C). The sites at which the of the RPA-mCherry signal revealed an apparent velocity of Sgs1 RPA-mCherry signal first appeared indicated that Sgs1 bound translocation of 80 ± 24 bp/s (n = 100) and a processivity of 7.8 ± randomly along the dsDNA (Fig. 1D) and then translocated in one 0.6 kb (n = 100) (Fig. 1 F and G). or both directions (Fig. 1E), similar to what has been reported for To visualize Sgs1 movement directly, we generated a GFP- bacterial RecQ (43). Specifically, the majority of events (73%; n = tagged version of Sgs1, expressed and purified it to near homo- 73 of 100) were associated with translocation in one direction only geneity, and then conducted biochemical assays to demonstrate (Fig. 1C), as schematically illustrated in Fig. 1B. The remaining that it possesses the wild-type level of DNA-dependent ATPase 27% (n = 27 of 100) of the initiating events led to Sgs1 trans- activity (SI Appendix,Fig.S1A). When examined on double- location in both directions; in these cases, it is likely that two (or tethered dsDNA curtains, GFP-Sgs1 unwound the dsDNA at

6092 | www.pnas.org/cgi/doi/10.1073/pnas.1819276116 Xue et al. Downloaded by guest on September 29, 2021 multiple locations, as evidenced by the binding of RPA-mCherry A B (Fig. 1H): in this case, 15.1% (n = 8 of 53) and 84.9% (n = 45 of barrier (B)

53) of the initiating events involving translocation of Sgs1 in both 3’ directions or in just one direction, respectively (Fig. 1H). GFP- Sgs1 exhibited a translocation velocity of 79 ± 27 bp/s and proc- flow dsDNA ssDNA essivity of 7.9 ± 0.6 kb (Fig. 1 I and J), being comparable to the values obtained for unlabeled Sgs1 (Fig. 1 F and G). Moreover, free DNA ends (F) GFP-Sgs1 exhibited similar velocity (87 ± 23 bp/s) and processivity B C GFP-Sgs1, +ATP 2 μm flow (8.7 ± 0.9 kb) in reactions with unlabeled RPA, indicating that the mCherry tag has no negative impact on the functionality of RPA (SI Appendix,Fig.S1B and C). Together, these findings reveal F that Sgs1 can initiate dsDNA unwinding from any internal loca- tion within the dsDNA molecule. D 15

10 Sgs1 Is Targeted to DSB Ends in the Absence of ATP. We next used

single-tethered dsDNA curtains to determine whether Sgs1 Count 5 would associate with DNA ends (Fig. 2A). Previous studies have shown that a short 3′ ssDNA tail, generated through the action 0 0 10 20 30 40 50 of MRX and Sae2, may provide the initial platform for Sgs1 Position (kbp) loading (26, 44, 45). To mimic this physiological substrate, we tested a λ-DNA substrate bearing a 30-nt 3′ ssDNA poly(dT) B E 2 μm overhang (Fig. 2B). Surprisingly, when GFP-Sgs1 (0.2 nM) was GFP-Sgs1, –ATP flow coinjected with ATP (2 mM), the protein did not associate preferentially with dsDNA ends, but instead bound to random F locations (Fig. 2 C and D), similar to what was observed in the 40 double-tethered assays. We next conducted experiments where F B flow GFP-Sgs1 was injected without ATP; for brevity, we will refer to 30 3 μm theATP-freeformofSgs1as“apo-Sgs1.” Importantly, the majority 1 min = 20

F BIOCHEMISTRY (76%) of apo GFP-Sgs1 molecules (n 183 of 242) colocalized with Count DNA ends (Fig. 2 E and F). Moreover, photobleaching step 10 measurements suggested that Sgs1 bound to the DNA ends as a 0 λ 010203040 50 dimer (Fig. 2 G and H). We also tested -DNA substrates with Position (kbp) either a 30-nt poly(dT) 5′ ssDNA overhang, a 12-nt 5′ ssDNA overhang, or a blunt dsDNA end. The 30-nt 5′ ssDNA overhang G 2 sec H 50 flow 100 also allowed for end recruitment of apo GFP-Sgs1, albeit at a 40 80 lower efficiency (∼50%, n = 101 of 203) compared with the 30-nt ′ 30 60 3 ssDNA overhang (SI Appendix,Fig.S2A). We were unable to 20 40 detect preferential association of GFP-Sgs1 with the shorter 12-nt Count 10 20 5′ ssDNA overhang (SI Appendix,Fig.S2B). Together, these re- Intensity (a.u.) 0 0 sults indicate that in the absence of ATP, Sgs1 is efficiently 0102030 1234 recruited to long (>12-nt) 5′ or 3′ ssDNA overhangs. Time (sec) # Steps Fig. 2. Sgs1 can be recruited to DNA ends through a Top3-Rmi1–independent DSB-Bound Sgs1 Cannot Initiate Long-Range Translocation. Our results pathway. (A) Schematic of single-tethered DNA curtain assay. The nomencla- demonstrated that Sgs1 is a highly active helicase (as revealed ture “B” and “F” are used to denote the ends of the DNA at the barrier and in the presence of RPA-mCherry) that can engage dsDNA and the free DNA ends, respectively, here and throughout. (B) Schematic of the unwind the DNA strands efficiently (Fig. 1). Interestingly, Sgs1 single-tethered dsDNA substrate with a 30-nt ssDNA 3′ overhang. (C)Wide- associates stably with DNA ends in its “apo” configuration (i.e., field image showing GFP-Sgs1 (green) bound randomly along the dsDNA in the absence of ATP) (Fig. 2 E and F). The nucleoprotein (unlabeled) when coinjected with 2 mM ATP. Note that buffer flow is turned complex of apo-Sgs1 with DNA ends is highly stable, exhibiting a ON in all single-tethered DNA curtain measurements and the direction flow is half-life of ∼19 min in the absence of ATP (see Fig. 4A, discussed from the top of the image to the bottom (as indicated), for this and all sub- below). Surprisingly, we saw no evidence for translocation of sequent kymographs from single-tethered experiments. (D) Binding distribu- end-bound GFP-Sgs1 even upon the addition of ATP (SI Ap- tion histogram of GFP-Sgs1 on dsDNA in ATP buffer (n = 259). (E) Wide-field pendix, Fig. S3A). Instead, the addition of ATP reduced the half- image showing that GFP-Sgs1 (green) is targeted to the DNA ends when ATP is life end-bound GFP-Sgs1 from ∼19 min to ∼12 min (see Fig. 4A, omitted from the buffer. (F) Binding distribution of GFP-Sgs1 on the DNA in discussed below). These results were striking in that, even though the absence of ATP (n = 242). The blue line represents as Gaussian fit to the Sgs1 is clearly a potent helicase, once bound to a DNA end, apo- data. (Inset) Kymograph showing that the end-bound GFP-Sgs1 (green) does Sgs1 remains in its inactive conformation for an extended period not exhibit evidence for translocation activity. (G) Graph showing a photo- of time and does not spontaneously convert to the active form of bleaching step trace for end-bound GFP-Sgs1. (Inset) Kymograph correspond- ing to the graphed data. Arrows highlight the photo-bleaching steps corre- the helicase. sponding to the bleaching of individual GFP monomers. (H) Distribution of photo-bleaching steps observed for end-bound GFP-Sgs1 (n = 151). Top3-Rmi1 Promotes Sgs1 Recruitment to DSBs Under Physiological Conditions. Previous studies have shown that Top3-Rmi1 stimulates DNA end resection by enhancing Sgs1activity(22,23).Tostudythe observed its binding to random locations along the dsDNA (Fig. role of Top3-Rmi1 in end resection, we first coinjected GFP- 3 A and B), similar to what was seen in the absence of Top3- Sgs1 and Top3-Rmi1 (0.2 nM each) in the absence of ATP, but under these conditions we were unable to detect any stable DNA Rmi1 (Fig. 2 C and D). binding for GFP-Sgs1. This result was in striking contrast to the Following the optimized biochemical conditions for Sgs1 end-specific binding activity observed when Top3-Rmi1 was absent helicase activity, the experiments described above were per- (Fig. 2 E and F). Next, we coinjected GFP-Sgs1 and Top3-Rmi1 formed under low salt conditions (i.e., 1 mM MgCl2 and no in the presence of ATP. Under these conditions, we found no monovalent salt) (23, 46). However, the stimulatory effect of evidence of GFP-Sgs1 recruitment to the DNA ends, but instead Top3-Rmi1 is most readily observed under physiological ionic

Xue et al. PNAS | March 26, 2019 | vol. 116 | no. 13 | 6093 Downloaded by guest on September 29, 2021 RPA Dislodges apo-Sgs1 from DNA Ends. We next asked whether the A GFP-Sgs1 + Top3-Rmi1 + ATP (low salt; minus RPA) B inclusion of RPA would affect the behavior of DNA end-bound

2 μm flow apo-Sgs1. Remarkably, addition of 2 nM RPA-mCherry (minus ATP) reduced the half-life of DNA end-bound GFP-Sgs1 from F ∼19 min to just ∼1.5 min (Fig. 4A), suggesting that RPA can dislodge prebound apo-Sgs1 from DNA ends. We could occa- 20 B sionally briefly detect RPA-mCherry and GFP-Sgs1 colocalization 15 at the DNA ends before Sgs1 dissociated (13.6%, n = 11 of 81) 10 (Fig. 4B). However, in most cases (86.4%; n = 70 of 81) the DNA

Counts 5 ends showed no detectable RPA-mCherry signal even though GFP-Sgs1 rapidly dissociated (Fig. 4B). The 30-nt 3′ ssDNA 0 λ 010203040 50 tail present on the -DNA used in these experiments should Position (kbp)

C GFP-Sgs1 + Top3-Rmi1 + ATP (high salt; minus RPA) B chased with buffer containing 2 μm flow A pre-bound GFP-Sgs1 ±ATP, ±RPA (as indicated)

1.0 F –ATP; t =19 ± 5 min, N=103 1/2

+ATP; t =12 ± 1 min, N=106 D 40 1/2 3 μm flow 0.5 30 1 min –ATP, +RPA; t =1.5 ± 0.2 min, N=68 1/2 20 +ATP, +RPA; t > 30 min, N=55

Counts 1/2

10 Survival probability 0 0 102030 0 Time (min) 010203040 50 Position (kbp) GFP- RPA- B Overlay Sgs1 mCherry Fig. 3. Top3-Rmi1–dependent recruitment of GFP-Sgs1 to DNA ends. (A) Wide-field image showing GFP-Sgs1 (green) coinjected with Top3-Rmi1 2 μm (unlabeled) bound randomly along the single-tethered dsDNA when coin- Sgs1 dissociates, 1 min N=70/81 (86.4%) no RPA signal jected with 2 mM ATP under low salt conditions (0 mM NaCl and 1 mM MgCl2). (B) Binding distribution of GFP-Sgs1 coinjected with Top3-Rmi1 Sgs1 dissociates, bound randomly along the DNA when coinjected with 2 mM ATP under detectable RPA N=11/81 (13.6%) low salt conditions (n = 224). (C) Wide-field image showing GFP-Sgs1 (green) signal coinjected with Top3-Rmi1 (unlabeled) and 2 mM ATP under physiological 0 0.2 0.4 0.6 0.8 salt conditions (100 mM NaCl and 5 mM MgCl2). (D) Binding distribution of GFP-Sgs1 coinjected with Top3-Rmi1 and ATP under physiological salt con- Fraction ditions (n = 189). (Inset) Kymograph showing that the end-bound GFP-Sgs1 CD (green) does not exhibit evidence for translocation activity. Note, there was GFP-Sgs1 GFP-Sgs1 no RPA included in these experiments. F 2 μm F 2 μm RPA-mCherry 2 min RPA- F strength (22, 23). Therefore, we conducted additional experi- F mCherry ments with 100 mM NaCl and 5 mM MgCl2 (Methods) to more Overlay F closely mimic the physiological setting. Under these conditions, Overlay we were unable to detect binding of GFP-Sgs1 alone to DNA F with ATP being present. However, upon the coinjection of GFP- +RPA, +ATP = Sgs1 and Top3-Rmi1, the majority (62%) of GFP-Sgs1 (n 117 EF100 of 189) was found at DNA ends (Fig. 3 C and D). The results thus revealed that Top3-Rmi1 promotes the ATP-dependent 30” 0” 10” 20” 50 DNA end recruitment of Sgs1 at physiological ionic strength. However, we still saw no evidence for GFP-Sgs1 translocation, RPA-mCherry which indicated that Top3-Rmi1 alone is insufficient for acti- intensity (a.u.) 0 40” 50” 60” 050100 vating DNA unwinding by end-bound Sgs1 (Fig. 3D, Inset). Time (s) Interestingly, in cells end resection can be initiated in the absence of the MRX complex at clean DSBs, but only after a Fig. 4. Effects of RPA of end-bound GFP-Sgs1. (A) Survival probability plot ∼1–2 h delay (17) and resection can even take place in showing the lifetimes of apo GFP-Sgs1 prebound to DNA ends when chased ± ± mre11 exo1 cells, albeit inefficiently (17). These findings suggest with either buffer containing 2 mM ATP and/or 2 nM RPA-mCherry. The solid lines represent single exponential fits to the data. (B) Kymographs the possibility that Sgs1 may be recruited to DSBs even in the showing the observed outcomes for end-bound apo GFP-Sgs1 upon the in- absence of MRX-generated overhangs. However, as indicated jection of RPA-mCherry and graph depicting the relative frequency of each above, we were unable to detect of GFP-Sgs1 recruitment to outcome. (C) Wide-field image showing the colocalization of end-bound blunt DNA ends under physiological salt conditions in the GFP-Sgs1 (green) and RPA-mCherry (magenta) upon coinjection of both presence of ATP (SI Appendix,Fig.S4A), nor did the addition 2 nM RPA-mCherry and 2 mM ATP. (D) Kymograph showing colocalization of of Top3-Rmi1 allow for the recruitment of GFP-Sgs1 to blunt GFP-Sgs1 (green) with RPA-mCherry (magenta). Arrow indicates the injection DNA ends (SI Appendix,Fig.S4B). These findings suggest that time point of RPA-mCherry and ATP. (E) Snapshots illustrating the accu- mulation and stability of RPA-mCherry (magenta) at DNA ends bound by some other low-level nuclease activity may be necessary to GFP-Sgs1 in the presence of ATP. (F) Quantification of the RPA-mCherry provide overhangs compatible for Sgs1 binding in mre11 exo1 fluorescence signal intensity (a.u.) for the molecule shown in E. Top3- mutant cells (17). Rmi1 was not present in any of the reactions shown in this figure.

6094 | www.pnas.org/cgi/doi/10.1073/pnas.1819276116 Xue et al. Downloaded by guest on September 29, 2021 ∼ be sufficient to allow binding 1 RPA-mCherry molecule (47). A GFP-Sgs1 (added 1st) D GFP-Sgs1 + Top3-Rmi1 (added 1st) Accordingly, control experiments performed in the absence of + Dna2 (added 2nd) + Dna2 (added 2nd) GFP-Sgs1 revealed that just 4.8% (n = 21 of 439) of the observed λ-DNA molecules exhibited RPA-mCherry signal (SI Appendix, flow Fig. S3B). Based upon these considerations, it is likely that the flow DNA ends had a single RPA-mCherry molecule associated with them, but the fluorescence signal might have fallen below the sensitivity limit of detection. 2 μm 2 μm 1 min 1 min RPA Plus ATP Activates Sgs1 for Short-Range Helicase Activity. As shown above, in the absence of ATP, RPA acts to dislodge apo- B 69 ± 21 bp/s E 93 ± 17 bp/s Sgs1 from DNA ends. In striking contrast, when RPA-mCherry 159 ± 40 bp/s 203 ± 21 bp/s (N=98) (N=200) (2 nM) was coinjected with 2 mM ATP, the vast majority of the 15 30 end-bound GFP-Sgs1 colocalized with RPA-mCherry (93%, n = 20 289 of 312) (Fig. 4 C and D). The GFP-Sgs1 signal was stable (t1/2 > 10 30 min) (Fig. 4A). Moreover, the RPA-mCherry was also stable Counts and its intensity increased gradually with time (Fig. 4 E and F), Count 5 10 indicative of RPA-mCherry accumulation at the DNA ends. Nev- ertheless, there was no evidence for detectable movement of GFP- 0 0 Sgs1 along the DNA (Fig. 4D); this observation again presents a 050100 150 200 250 0 100 200 300 striking contrast to the extensive dsDNA unwinding activity ob- Velocity (bp/s) Velocity (bp/s) served when Sgs1 was coinjected with ATP (i.e., Fig. 1). Similarly, C 16.5 ± 4.1 kb F control experiments conducted with 2 mM ATP and 2 nM un- 1.0 1.0 27.8 ± 2.6 kb labeled RPA also failed to reveal evidence for long-range move- 11.1±1.7 kb (N=98) ment of end-bound GFP-Sgs1 along the DNA (SI Appendix,Fig. 19.1±2.1 kb S3C). We could conclude that coaddition of ATP and RPA may (N=200) have enabled Sgs1 to unwind a short portion of DNA from ends, 0.5 0.5 which would allow for more extensive RPA binding, but would still

be insufficient to convert Sgs1into its fully activated state. BIOCHEMISTRY Survival Probability Dna2 Activates DNA End-Bound Sgs1 for Long-Range Translocation. 0 Survival Probability 0 Sgs1 partners with Dna2 in DNA end-resection (21–23). There- 01020 30 4050 01020 30 4050 Distance (kbp) fore, we next asked whether Dna2 is able to convert apo GFP- Distance (kbp) Sgs1 to its active state in the presence of ATP. Remarkably, Dna2 Fig. 5. Dna2 activates long-distance translocation by end-bound Sgs1. (A) allowed for long-range translocation of end-bound GFP-Sgs1 even Kymograph showing that GFP-Sgs1 (green) prebound to DNA ends un- when Top3-Rmi1 was absent (Fig. 5A). Note, that in these assays dergoes long-distance translocation (in the absence of Top3-Rmi1) upon GFP-Sgs1 is prebound to the DNA ends and the unbound pro- coinjection of 0.2 nM Dna2 and 2 mM ATP. (B) Velocity distribution of end- teins are then flushed from the sample chamber. Therefore, the bound GFP-Sgs1 upon coinjection of 0.2 nM Dna2 and 2 mM ATP. The ma- distance that the end-bound GFP-Sgs1 travels upon activation by genta and green lines represent Gaussian fits to the data for the slower and Dna2 is a direct reflection of the processivity of Sgs1. A nuclease- faster populations, respectively. (C) Survival probability plots showing the deficient Dna2 mutant (D657A) (22) and a helicase-deficient distance that end-bound GFP-Sgs1 translocated upon coinjection of 0.2 nM Dna2 mutant (K1080E) (48) similarly activated long-range trans- Dna2 and 2 mM ATP. The data were divided into two populations, based upon location of end-bound GFP-Sgs1 (SI Appendix, Fig. S5), indicating whether the complexes belong to the slower (magenta) or faster (green) that neither the nuclease nor the helicase activity of Dna2 was subpopulations shown in B.(D) Kymograph showing GFP-Sgs1 (green) pre- required for Sgs1 activation. Surprisingly, analysis of the resulting bound to dsDNA ends in the presence Top3-Rmi1 was activated for long-range trajectories yielded bimodal velocity and processivity distributions, translocation upon subsequent coinjection of 0.2 nM Dna2 plus 2 mM ATP. (E) suggesting the existence of two subpopulations of activated Sgs1– Velocity distribution of Top3-Rmi1 loaded GFP-Sgs1 (green) in reactions chased Dna2 complexes (Fig. 5 B and C and SI Appendix, Table S1). One with 0.2 nM Dna2 and 2 mM ATP. The magenta and green lines represent = Gaussian fits to the data for the slower and faster populations, respectively. (F) population, representing 24.5% of the observed molecules (n Survival probability plots showing the distance that end-bound GFP-Sgs1 24 of 98), exhibited a velocity of 69 ± 21 bp/s and a processivity of ± translocated upon coinjection of 0.2 nM Dna2 plus 2 mM ATP. Data were di- 16.5 4.1 kb (Fig. 5 B and C). The second population, repre- vided into two populations, based upon whether the complexes belong to the senting 75.5% of the observed molecules (n = 74 of 98), exhibited slower (magenta) or faster (green) subpopulations shown in B. velocity and processivity values of 159 ± 40 bp/s and 27.8 ± 2.6 kb (n = 98), respectively (Fig. 5 B and C). We note that these Dna2- dependent Sgs1 translocation velocity and processivity values injection of Dna2 (0.2 nM). These results showed that Dna2 exceeded those obtained for Sgs1 when coinjected with RPA and activates the long-range translocation activity of the end-bound ATP by 1.8- and 3.3-fold, respectively (compare Figs. 1F and 5 B GFP-Sgs1–Top3-Rmi1 complex (Fig. 5D). Data analysis again and C). Together, our results reveal that Dna2 converts inactive revealed two distinct populations, with one population (n = 93 of DNA end-bound GFP-Sgs1 from its apo state into a highly active 200 or 46.5% of the molecules observed) exhibiting translocation conformation capable of translocating thousands of base pairs and processivity values of 93 ± 17 bp/s and 11.1 ± 1.7 kb, respec- along the dsDNA. tively, and the other (n = 107 of 200 or 53.5% of the molecules observed) possessing translocation and processivity values of 203 ± Cooperation Between Dna2 and Top3-Rmi1 in Sgs1 Activation. Even ± though Top3-Rmi1 promotes the recruitment of GFP-Sgs1 to 21 bp/s and 19.1 2.1 kb, respectively (Fig. 5 E and F). In- DNA ends in the presence of ATP at physiological ionic terestingly, the processivity values for both subpopulations were strength, GFP-Sgs1 remains tightly bound to the DNA ends and slightly lower than those observed in the absence of Top3-Rmi1 does not appear to move (Fig. 3 C and D). We asked whether (compare Fig. 5 C and F). In contrast, the translocation velocities Dna2 might allow for activation of GFP-Sgs1 under these cir- of both subpopulations increased by ∼35% and ∼28%, respec- cumstances. For this, GFP-Sgs1 was prebound to free DNA ends tively, compared with reactions lacking Top3-Rmi1 (compare Fig. by coinjection of GFP-Sgs1 and Top3-Rmi1 (0.2 nM each) in 5 B and E), suggesting that Dna2 and Top3-Rmi1 act together to buffer containing 2 mM ATP and 100 mM NaCl, followed by the enhance translocation of GFP-Sgs1.

Xue et al. PNAS | March 26, 2019 | vol. 116 | no. 13 | 6095 Downloaded by guest on September 29, 2021 RPA Is Necessary for DNA End Processing. The DNA intercalating A GFP-Sgs1 (pre-bound) Dna2 + RPA-mCherry (added 2nd) dye YoYo1 emits a bright fluorescent signal when bound to (+YoYo1, present throughout) dsDNA but has a lower affinity for ssDNA. This dye has been used 40 1.0 4 μm 50 ± 18 bp/s extensively to monitor end resection in single-molecule assays with YoYo1 2 min (N=86) the Escherichia coli RecBCD complex (49–52). We next con- 30 ducted DNA curtain to assays in the presence of YoYo1 to de- flow 9.3 ± 0.5 kb 20 0.5 termine whether cofactor-mediated activation of DNA end-bound RPA- (N=86) mCherry Count Sgs1 coincides with DNA resection. Specifically, DNA resection 10 would be revealed by the loss of YoYo1 signal intensity coinciding Survival Probability 0 Overlay 0 with the appearance of RPA-mCherry on ssDNA overhangs 0 100 200 01020 30 4050 generated by end resection. Velocity (bp/s) Distance (kbp) Consistent with previous results (22, 23), control experiments confirmed that Dna2 alone, or in combination with RPA and Top3-Rmi1 did not detectably degrade the single-tethered DNA B GFP-Sgs1 (pre-bound) Dna2 + RPA-mCherry (added 2nd) molecules (SI Appendix, Fig. S6 A–C). Moreover, there was no (–YoYo1, absent throughout) evidence for DNA resection based upon the YoYo1 signal in 30 107 ± 46 bp/s GFP 1.0 reactions with end-bound Sgs1 when chased with buffer containing -Sgs1 (N=133) 10.7 ± 1.2 kb 25

ATP and Dna2 (SI Appendix,Fig.S6D), which is also consistent flow (N=133) with previous studies (22, 23). These results indicate that even 20 RPA- 0.5 though end-bound Sgs1 exhibits rapid and highly processive mCherry 10 translocation upon Dna2 addition, the protein complex lacks re- Count section capability (Fig. 5 A–C). Moreover, we were unable to find 5 Overlay evidence of DNA resection in reactions containing Sgs1, Top3- 0 Survival Probability 0 0 100 200 01020 30 4050 Rmi1, Dna2, and ATP, but no RPA (SI Appendix,Fig.S6E), even Velocity (bp/s) Distance (kbp) though GFP-Sgs1 is otherwise highly processive in its movement along DNA under these conditions (Fig. 5 D–F). Thus, while complexes that harbor Sgs1, Top3-Rmi1, and Dna2 exhibit robust C GFP-Sgs1 + Top3-Rmi1 (pre-bound) Dna2 + RPA-mCherry (added 2nd) translocase activity, our results suggest that this attribute is decoupled (+YoYo1, present throughout) from end processing when RPA is absent from the reactions. Alter- 25 1.0 45 ± 31 bp/s natively, we cannot rule out the possibility that in the absence of RPA, YoYo1 Sgs1-dependent translocation can take place with not corresponding 20 (N=93) 7.3 ± 0.5 kb

flow (N=93) separation of the DNA strands. 15 0.5 In striking contrast, when RPA-mCherry was included in the RPA- 10 reactions with end-bound GFP-Sgs1, Top3-Rmi1, and Dna2, mCherry Count 5 28% of the observed dsDNA molecules (n = 68 of 319) exhibited the progressive loss of YoYo1 fluorescence signal emanating Overlay 0 Survival Probability 0 0100200 01020 30 4050 from DNA ends (Fig. 6A and SI Appendix, Fig. S6F). The ob- Velocity (bp/s) Distance (kbp) served loss of YoYo1 signal intensity likely stemmed from RPA activating DNA end resection by Sgs1–Dna2. Notably, the mCherry signal colocalized with DNA ends being acted on by D GFP-Sgs1 + Top3-Rmi1 (pre-bound) Dna2 + RPA-mCherry (added 2nd) Sgs1–Dna2 (Fig. 6A), providing strong evidence for the presence (–YoYo1, absent throughout) of a ssDNA tail, the expected product of resection, at these ends. 25 1.0 80 ± 37 bp/s In reactions with 2 nM RPA-mCherry, the mCherry signal did GFP 20 (N=104) not increase substantially with time, as would be expected if the -Sgs1 9.6 ± 1.1 kb

flow 15 (N=104) resection reactions yielded progressively longer ssDNA ends, 0.5 suggesting that the overall length of the ssDNA ends may be RPA- 10

mCherry Counts limited by Dna2 cleavage of both strands under these reaction 5 conditions (Fig. 6A). However, the mCherry signal did increase 0 Survival Probability 0 significantly when the concentration of RPA-mCherry was in- Overlay 0100200 01020 30 4050 creased 10-fold (20 nM), indicative of extensive ssDNA pro- Velocity (bp/s) Distance (kbp) duction, consistent with previous biochemical studies showing that RPA protects the 3′ strand from Dna2 nuclease degradation Fig. 6. RPA couples the translocation and nuclease activities of the end- (22, 23). Analysis of Sgs1–Dna2 translocation with RPA present resection machinery. (A) Kymograph showing the resection of YoYo1-stained revealed a velocity of 50 ± 18 bp/s and an average processivity of dsDNA (green) in the presence of prebound GFP-Sgs1 (not visible) when 9.3 ± 0.5 kb (Fig. 6A). We were unable to visualize GFP-Sgs1 at chased with Dna2, RPA-mCherry (magenta), and 2 mM ATP (Left)withthe the DNA ends because YoYo1 emitted a much stronger fluo- corresponding velocity distribution (Center) and processivity plot (Right). (Scale bar applies to all panels.) (B) Kymograph showing the movement of GFP-Sgs1 rescence signal, and we therefore conducted complementary (green) prebound to unlabeled dsDNA when chased with Dna2, RPA-mCherry measurements in the absence of YoYo1 (Fig. 6B). These results (magenta), and 2 mM ATP (Left) with the corresponding velocity distribution confirmed that GFP-Sgs1 and RPA-mCherry colocalized with (Center) and processivity plot (Right). (C) Kymograph showing the resection of DNA ends during resection (Fig. 6B). Interestingly, the trans- YoYo1-stained dsDNA (green) in the presence of prebound GFP-Sgs1 (not location velocity observed in the presence of YoYo1 was sig- visible) plus Top3-Rmi1, when chased with Dna2, RPA-mCherry, and 2 mM ATP nificantly slower than that observed in its absence (50 ± 18 bp/s (Left) with the corresponding velocity distribution (Center) and processivity vs. 107 ± 46 bp/s) (Fig. 6 A and B), which likely stemmed from an plot (Right). (D) Kymograph showing the movement of prebound GFP-Sgs1 inhibitory effect of YoYo1 on resection. (green) and Top3-Rmi1 when chased with on unlabeled dsDNA in the presence We next examined the behavior of GFP-Sgs1 recruited to DNA of Dna2, RPA-mCherry (magenta) and 2 mM ATP (Left) with the corresponding ends by Top3-Rmi1 and then activated for translocation and DNA velocity distribution (Center) and processivity plot (Right). endresectionbyDna2,RPA,andATP.First,weexaminedre- section with GFP-Sgs1, Dna2, Top3-Rmi1, and RPA using YoYo1- stained DNA and observed progressive loss of YoYo1 fluorescence velocity of 45 ± 31 bp/s and processivity of 7.3 ± 0.5 kb (Fig. 6C). signal with coincident RPA-mCherry binding for 23% (n = 49 of Experiments conducted in the absence of YoYo1 confirmed that 211) of DNA molecules (Fig. 6C). Analysis of data revealed a GFP-Sgs1 and RPA-mCherry colocalized with the DNA during

6096 | www.pnas.org/cgi/doi/10.1073/pnas.1819276116 Xue et al. Downloaded by guest on September 29, 2021 resection, and that the reaction with YoYo1 free DNA proceeded A Atto565-Nuc with a velocity that was approximately twice as fast (Fig. 6D). GFP-Sgs1 (low density) overlay Taken together, and consistent with published reports (22, 23), our B single-molecule analysis revealed a key function of RPA in acti- vating the DNA resection activity of Sgs1–Dna2.

3 μm Repositioning of Nucleosomes by the DNA End-Processing Machinery. F 1 min We have only rudimentary understanding of the impact of chro- matin structure on DNA end resection and of nucleosome dy- 30 BC1.0 namics therein (17, 19, 53, 54). To explore the relationship between end resection and chromatin, we next sought to de- 7.4 ± 1.2 kb 65 ± 30 bp/s 20 (N = 96) termine the impact of nucleosomes on resection catalyzed by the (N = 96) Sgs1–Dna2–Top3-Rmi1–RPA ensemble. For these experiments, 0.5 nucleosomes were fluorescently labeled with Atto565 using the Count 10 cysteine mutant (S47C) of histone H2A (55). The nucleosomal dsDNA substrate was prepared by salt dialysis, under conditions that yielded low-, medium-, or high- 0 Survival Probability 0 density nucleosome arrays (Methods). The low-density nucleo- 0 50 100 150 200 250 01020 30 4050 somal arrays had an average of ∼four nucleosomes per dsDNA Velocity (bp/s) Distance (kbp) molecule based upon visual inspection. Importantly, Sgs1– – D Atto565-Nuc Dna2 RPA readily translocated along the low-density nucleosome- GFP-Sgs1 (medium density) overlay bound dsDNA (Fig. 7A). Moreover, activated GFP-Sgs1 was B able to push nucleosomes over long distances along the dsDNA (Fig. 7A). The translocation velocity of GFP-Sgs1– Dna2–Top3-Rmi1–RPA was 65 ± 30 bp/s (Fig. 7B), corre- F sponding to an ∼19% reduction (Student t test, P < 0.05) 2 μm relative to that measured for naked DNA (80 ± 37 bp/s) (Fig. 2 min 6D). The mean processivity of GFP-Sgs1–Dna2–RPA was 7.4 ±

EF20 1.0 1.2 kb in the presence of nucleosomes (Fig. 7C), corresponding BIOCHEMISTRY ∼ < 7.8 ± 1.3 kb to a 23% reduction (Student t test, P 0.001) relative to naked 15 (N = 52) DNA (9.6 ± 1.1 kb) (Fig. 6D). We were unable to count the numbers of nucleosomes bound to 41 ± 22 bp/s each dsDNA under the medium- and high-density reconstitution 10 (N = 52) 0.5

Count conditions because the nucleosomes were too closely spaced on the 5 dsDNA (Fig. 7 D and G). For medium-density conditions, the dsDNA molecules appeared shortened due to nucleosome-induced ± μ Survival Probability DNA compaction, yielding an average length of 9.7 1.4 m 0 0 ± μ 0 50 100 150 200 250 01020 30 4050 compared with 12.5 0.2 m for naked DNA (at a buffer flow rate Velocity (bp/s) Distance (kbp) of 0.15 mL/min). Importantly, targeting of GFP-Sgs1 the ends of the nucleosome-bound DNA molecules occurred normally, and G Atto565-Nuc DNA translocation of the end-associated Sgs1 commenced upon GFP-RPA (high density) overlay the inclusion of Dna2, RPA, and ATP (Fig. 7D). However, the translocation velocity was reduced to just 41 ± 22 bp/s, although B the processivity of translocation remained quite high at 7.8 ± 1.3 kb (Fig. 7 E and F). F 1 μm At higher nucleosome density, the DNA exhibited a length 1 min of ≤2 μm, corresponding to ∼6.25-fold compaction relative to naked DNA. Even though GFP-Sgs1 could still associate with HI6 1.0 the ends in these DNA molecules, we were unable to visualize 479 ± 361 a.u. GFP-Sgs1 translocation due to the highly compacted nature of (N = 41) low, –SDTRR 4 * the substrate (Fig. 7G). As an alternative approach, we moni- low, +SDTRR tored the accumulation of RPA-GFP at DNA ends as a readout 0.5 for DNA resection; GFP-tagged RPA was used here to take Count 2 medium, +SDTRR advantage of the brighter and more stable fluorescence signal of GFP relative to mCherry. Indeed, we could detect a strong, time-

Intensity (a.u.) high, +SDTRR dependent accumulation of RPA-GFP signal upon inclusion of 0 0 0 2.5 5 7.5 10 the full complement of end-resection proteins, reflective of 0 300 600 900 1200 ssDNA being generated via end resection (Fig. 7G). To estimate RPA-GFP Intensity (a.u.) Time (min)

Fig. 7. Sgs1-dependent nucleosome mobilization during DNA end re- section. (A) Kymograph showing movement of GFP-Sgs1 (green) in the (F) Processivity for GFP-Sgs1 movement on medium density nucleosome- presence of Top3-Rmi1, Dna2, RPA, and 2 mM ATP on dsDNA bound by low bound DNA (as shown in D). (G) Kymograph showing the appearance of density Atto565-labeled nucleosomes (Atto565-nuc; shown in magenta). RPA-GFP (green) by the movement of Sgs1 in the presence of Top3-Rmi1, (B) Velocity distribution of GFP-Sgs1 on low-density nucleosome-bound Dna2, RPA-GFP, and 2 mM ATP on dsDNA bound by high-density Atto565- DNA (as shown in A). The blue line represents a Gaussian fit to the data. nucleosomes (magenta). (H) Distribution of RPA-GFP intensity values obtained (C) Processivity plot for GFP-Sgs1 movement on low-density nucleosome- from experiments conducted with high-density Atto565-nucleosomes (ma- bound DNA (as shown in A). (D) Kymograph showing the movement of genta). The asterisk highlights the signal intensity value expected for ∼one GFP-Sgs1 (green) in the presence of Top3-Rmi1, Dna2, RPA, and 2 mM ATP molecule of RPA-GFP. (I) Integrated signal intensity of Atto565-tagged on dsDNA bound by medium-density Atto565-nucleosomes (magenta). (E) nucleosomes for the low-, medium-, and high-density nucleosome arrays Velocity distribution of GFP-Sgs1 movement on medium-density nucleosome- during the course of the end resection assays ±SDTRR (Sgs1, Dna2, Top3– bound DNA (as shown in D). The blue line represents a Gaussian fit to the data. Rmi1, and RPA).

Xue et al. PNAS | March 26, 2019 | vol. 116 | no. 13 | 6097 Downloaded by guest on September 29, 2021 the length of ssDNA produced as a result of end resection, we indiscriminant binding DSB end 3’ low salt +Sgs1 MRX Top3-Rmi1 Dna2 measured the intensity of the RPA-GFP foci and then compared +ATP 3’ no binding Sgs1 RPA the values to the intensity of a single GFP molecule (Fig. 7H). We 3’ high salt found that Sgs1–Dna2–Top3-Rmi1 allowed for the binding of 8 ± Sgs1 loading

6 molecules of RPA-GFP, and given the ssDNA binding site +apo Sgs1 only +Sgs1, +Top3-Rmi1, +ATP ∼ (low salt) (high salt) size of RPA being 30 nt (47), reflecting the production of a Top3- ± limited unwinding +ATP Rmi1 +ATP limited unwinding 240 180 nt of ssDNA (Fig. 7H). Notably, we did not find any +RPA +RPA evidence of a significant loss of nucleosome fluorescence signal 3’ 3’ 3’ 3’ regardless of the nucleosome occupancy (Fig. 7I), suggesting +RPA Sgs1 activation Sgs1 activation that end resection is not accompanied by nucleosome eviction. 3’ +ATP +Dna2 Taken together, and consistent with published biochemical data +ATP –RPA +Dna2 – – – faster population (56), our results provide evidence that Sgs1 Dna2 Top3-Rmi1 –RPA 3’ faster population translocation, 3’ RPA is able to reposition nucleosomes while processing DNA, no processing but a high nucleosome occupancy presents a strong impediment to translocation, + + no processing slower population long-range resection. 3’ slower population translocation, 3’ no processing translocation, Sgs1 Possesses Intrinsic Nucleosome Remodeling Activity. no processing +ATP To ask +ATP Dna2 activation +Dna2 Dna2 activation whether Sgs1 alone is able to reposition nucleosomes, we performed +Dna2 +RPA +RPA translocation + translocation + experiments with double-tethered dsDNA curtains bound by a low end processing end processing density of Atto565-labeled nucleosomes. Under these conditions, 3’ 3’ we could readily observe GFP-Sgs1 colliding with individual nucle-

osomes (SI Appendix,Fig.S7A), followed by repositioning of nu- Top3-Rmi1-independent Top3-Rmi1-dependent cleosomes along the path of Sgs1 translocation at a mean velocity of 62 ± 13 bp/s for an average distance of 5.9 ± 0.5 kb (SI Appendix, ∼ ∼ Fig. 8. Model describing the loading and regulation of the Sgs1-dependent Fig. S7 B and C). Notably, these values were 20% and 25% DNA end processing machinery. See Discussion for details of the model. lower than the velocity and processivity, respectively, observed for GFP-Sgs1 translocation on naked dsDNA. We conclude that indi- vidual nucleosomes are readily mobilized as a result of ATP- apo-Sgs1 from a recessed DNA end, it could conceivably help dependent Sgs1 translocation. ensure the timely removal of apo-Sgs1 complexes that cannot be promptly activated by ATP and protein cofactors (Fig. 8). Discussion Interestingly, we have found that Top3-Rmi1 serves to target DNA end resection represents the critical decision point regarding Sgs1 to recessed DNA ends when ATP is present, but only at whether a DSB will be repaired through mostly error-free path- physiological salt concentrations; given that ATP is always pre- ways of HR, or by the error-prone nonhomologous end-joining or sent in cell, this Top3-Rmi1–dependent pathway mostly likely a microhomology-mediated DNA end-joining mechanism (17, 19, mimics the biologically relevant mechanism (Fig. 8). Top3- 20). Here, we have used DNA curtain assays to examine dsDNA Rmi1 does not increase the velocity or processivity of the end- end resection at the single-molecule level, providing insights into resection machinery; indeed, its presence slows the translocation the functional interplay among end-resection proteins. Our results velocity of GFP-Sgs1 and reduces processivity (Fig. 6 A–D). In- are germane for understanding the end recruitment and activation stead, the primary effect of Top3-Rmi1 is that it drastically in- of the Sgs1-dependent DNA end-resection machinery. Our find- creases Sgs1 recruitment to DNA ends in reactions containing ings with the yeast end-resection factors may have direct bearing ATP (compare Figs. 2C and 3C). This finding suggests that the on the equivalent mechanisms of BLM-dependent end resection stimulatory effect of Top3-Rmi1 observed in bulk assays may be in higher eukaryotes. attributed to enhance Sgs1 end recruitment. Thus, our findings are most consistent with a model where Top3-Rmi1 promotes Two Pathways for End Recruitment of Sgs1. Our work shows that recruitment of Sgs1 to MRX-processed DNA ends. Remarkably, Sgs1 can be recruited to DSB ends through either a Top3-Rmi1– the end-bound Sgs1–Top3-Rmi1 complex also remains station- independent mechanism or a Top3-Rmi1–dependent mechanism ary, even when ATP is present, implying that Sgs1 is still main- (Fig. 8). Specifically, Sgs1 in its apo configuration and at low tained in its inactive state with regards to DNA translocation and ionic strength is efficiently targeted to DNA ends bearing a 3′ unwinding. Interestingly, Top3-Rmi1 also prevents the re- ssDNA overhang that mimics the earliest resection intermediate cruitment of apo-Sgs1 (i.e., when ATP is absent) to DNA ends. generated by the MRX/Sae2-catalyzed endonucleolytic and We surmise that either Top3-Rmi1 binds DNA ends at the ex- subsequent 3′ → 5′ exonucleolytic processing of DSBs (Fig. 8). pense of apo-Sgs1 engagement or interaction of Top3-Rmi1 with Thus, omission of ATP from these reactions allows for highly apo-Sgs1 prevents their end association. Therefore, we have selective end-binding by Sgs1 in the absence of Top3-Rmi1. We provided evidence that Top3-Rmi1 and RPA can both prevent do not expect that reactions lacking ATP mimic cellular condi- apo-Sgs1 from accumulating at DNA ends, possibly through tions given that ATP is highly abundant in vivo. However, re- distinct mechanisms (Fig. 8). actions lacking ATP provide an essential comparison for the Our data show that Sgs1 can be targeted to DNA ends only reactions that do contain ATP. Moreover, we speculate that the under specific conditions: either low salt buffer in the absence of omission of ATP may mimic some aspects of the normal Top2- ATP or in physiological salt buffer containing ATP and Top3- Rmi1–dependent Sgs1 recruitment mechanism: for example, by Rmi1 (Fig. 8). In either scenario, end targeting may occur either enabling Sgs1 to adopt a conformation with little affinity for through: (i) a reduced affinity of Sgs1 for internal sites on the dsDNA—thus preventing its association with DNA internally— dsDNA, (ii) through an increased affinity of Sgs1 for the ssDNA but one that is capable of binding a ssDNA overhang or dsDNA/ (or ssDNA/dsDNA junctions) present at the ends of the sub- ssDNA junctions avidly. Importantly, this end-bound Sgs1, which strates, or (iii) through a combination of these two affects. Our we have termed “apo” Sgs1, cannot be activated by ATP, even present study does not distinguish between these possibilities. though, based on results from additional analyses (compare Fig. 5 and SI Appendix, Fig. S3A) (see discussion below) the catalytic Activation of DNA End-Bound Sgs1 by Dna2. Sgs1 can be targeted to potential for DNA translocation and DNA unwinding is clearly DNA ends either under low salt conditions in the absence of preserved within apo-Sgs1. Thus, dsDNA binding, DNA end ATP, or under more physiological salt conditions in the presence engagement, and DNA translocation/unwinding are separable of Top3-Rmi1 and ATP. However, Sgs1 remains catalytically attributes of Sgs1. Because RPA provokes rapid dissociation of dormant in both scenarios. Importantly, we have shown that

6098 | www.pnas.org/cgi/doi/10.1073/pnas.1819276116 Xue et al. Downloaded by guest on September 29, 2021 Dna2 activates end-bound Sgs1 for long-range translocation with Notably, the rate of DNA end resection mediated by the full and without Top3-Rmi1 being present (Fig. 8). Consistent with complement of Sgs1, Dna2, Top3-Rmi1, and RPA in our assays previous reports, neither the ATPase nor nuclease activity of is ∼80 bp/s. In contrast, in S. cerevisiae and Schizosaccharomyces Dna2 is required for Sgs1 activation (22, 23). These findings pombe cells, the resection of DSBs proceeds at ∼4–6 kb/h, cor- indicate that activation of long-range translocation and DNA responding to a resection rate of just ∼1.1–1.7 bp/s (21, 64, 65). unwinding is tightly coupled to the full assembly of the end- These observations strongly suggest that there are additional resection machinery and reveal a critical licensing role of factors, such as higher-order chromatin structure, that restrain Dna2 in this regard (Fig. 8). the rate and extent of end resection in living cells. Notably, there is an ∼30% increase in Sgs1 translocation ve- locity when Dna2-mediated activation occurs through the Top3- Methods Rmi1–dependent loading mechanism, thus providing evidence End-Resection Proteins. S. cerevisiae RPA, RPA-mCherry, RPA-GFP, Dna2, and for a cooperative effect between Dna2 and Top3-Rmi1 in Sgs1 Top3-Rmi1 were all expressed and purified as described previously (22, 66, activation. However, long-range translocation of complexes of 67). Full-length Sgs1, labeled at the N terminus with His6 and FLAG tags and Sgs1–Dna2 is not accompanied by significant DNA end resection labeled at the C terminus with a HA tag, was expressed and purified form or DNA unwinding (Fig. 8). Given that Sgs1 alone is a robust Sf9 insect cells, as previously described (22). GFP-tagged Sgs1 was prepared dsDNA helicase (46), we infer that the Sgs1–Dna2 complex by inserting the GFP gene between the His6-FLAG tags and the N terminus separates DNA strands while translocating on dsDNA (in the of the Sgs1 gene in a pFastBac-HTB vector (Invitrogen). A bacmid was then absence of RPA); however, rapid reannealing of the unwound generated in the E. coli strain DH10Bac (Invitrogen), and the GFP-tagged strands behind the translocating protein complex, as evidenced Sgs1 protein was expressed in insect cells, as described for the unlabeled by YoYo1 staining, would greatly limit the extent of ssDNA protein (22). Details of Sgs1 ATP hydrolysis assays, DNA substrate construc- region that can accumulate (Fig. 8). However, we cannot com- tion, histone purification, and nucleosome reconstitution are provided in SI Appendix. pletely rule out the possibility that in the absence of RPA, the Sgs1–Dna2 can also undergo translocation on dsDNA without Single-Molecule Assays. Experiments were conducted with a custom-built separating the dsDNA strands. Finally, although we favor a prism-type TIRF microscope (Nikon) equipped with a 488-nm laser (Co- model where Dna2 (and Top3-Rmi1) tracks with Sgs1 as part of herent Sapphire, 200 mW) and a 561-nm laser (Coherent Sapphire, 200 mW). the STR complex during translocation and end processing, be- All single-molecule experiments were performed at 30 °C in resection buffer

cause we do not have labeled Dna2 (or Top3-Rmi1), we cannot [20 mM Tris·HCl (pH 7.5), 1 mM MgCl2, 1 mM DTT, 0.2 mg/mL BSA; ±2mM yet directly verify this possibility. ATP and ±0.5 nM YoYo1, as indicated]. Double-tethered and single-tethered

dsDNA curtains were prepared as described previously (42, 66, 68). For BIOCHEMISTRY RPA Couples ATP-Dependent DNA Translocation to End Processing. double-tethered DNA curtain assays, there was no buffer flow during data RPA exerts multiple effects on end-bound Sgs1. First, when ATP acquisition. In contrast, for all single-tethered assays, buffer flow was always is present, RPA appears to activate Sgs1 for limited short-range on during data collection, and the flow direction in all kymographs was translocation, as evidenced by the stable colocalization of RPA always from the top to the bottom of the images, as indicated in the rele- and Sgs1 at DNA ends (Fig. 8). Second, upon Sgs1 activation by vant figure panels. The sample chambers were flushed with reaction buffer, Dna2, the resulting complex displays a bimodal distribution of and reactions were initiated by injecting 150 or 500 μL Sgs1 (0.2 nM), GFP- translocation velocities with or without Top3-Rmi1 being pre- Sgs1 (0.2 nM), ±0.2 nM Top3-Rmi1 (as indicated) in Sgs1 reaction buffer at a flow rate of either 0.05 mL/min or 0.15 mL/min. Unbound proteins were sent, and similar results were obtained for the nuclease- and – helicase-deficient mutants of Dna2. These findings suggest that flushed away by washing with 2 5 mL Sgs1 reaction buffer at 0.5 mL/min. the Sgs1–Dna2 complex exists in two distinct conformations and DNA end resection was initiated by injecting 1 mL indicated protein (0.2 nM Dna2, 2 nM RPA or RPA-mCherry, or RPA-GFP) at 0.15 mL/min. Image ac- stoichiometries, manifested as populations exhibiting translocation ∼ – ∼ – quisition was started immediately before the protein injections. All images velocity of 160 170 bp/s or 70 80 bp/s (Fig. 8). Remarkably, the were acquired at one frame per 10 s with 0.1-s integration time using two addition of RPA led to the assembly of a single class of the Sgs1– ∼ EMCCD cameras, and the illumination lasers were shuttered between each Dna2 complex with a translocation velocity of 100 bp/s (Fig. 8). image to minimize photo-bleaching (66). Importantly, consistent with resultsfrombiochemicalreconstitution studies (22, 23), RPA proves to be essential for triggering nucleo- Imaging Processing and Data Analysis. Raw TIFF images were imported as lytic end processing by the Sgs1–Dna2 complex (Fig. 8). Thus, RPA image stacks into ImageJ. Images were corrected for drift using the StackReg is crucial for coupling the translocation of activated Sgs1 to the function in ImageJ. Kymographs were then generated from the corrected DNA end-processing activity of Dna2. image stacks by defining a one-pixel wide region-of-interest encompassing individual dsDNA molecules, and these kymographs were used for analysis of The Influence of Chromatin on Sgs1-Dependent End Resection. A GFP-Sgs1 processivity and velocity (66). The total length of the λ-DNA sub- number of ATP-dependent chromatin remodelers, including strate is 48,502 bp. The DNA was extended to a mean length of 12.5 ± 0.2 μm RSC, SWI/SNF, INO80, SWR-C, and Fun30, are recruited to at a flow rate of 0.15 mL/min and each DNA spanned a distance of ∼47 pixels DSBs in S. cerevisiae (57–63). However, biochemical studies have at 60× magnification. In this case, the measured-length pixels were con- provided convincing evidence that the Sgs1-dependent end- sidered as 1,060 bp of DNA per pixel. Sgs1 translocation events from the resection machinery has an intrinsic ability to remodel nucleo- kymographs were fitted with a linear function to calculate translocation somes and is thus less prone to the presence of nucleosomes on velocity for each individual molecule. Translocation velocities then obtained from Gaussian fits of the distributions of observed velocities. The the DNA template than does the Exo1-mediated DNA resection distance of translocation events was used to generate survival probability mechanism (56). Our single-molecule analysis provides direct vi- plots and the reported processivity values reflect the translocation dis- sual evidence for the role of the Sgs1 ATP hydrolysis-dependent tance at which one-half of Sgs1 stops translocation along the DNA. Unless DNA motor activity in promoting nucleosome mobilization. Im- otherwise stated, error bars represent 95% confidence intervals calculated portantly, this activity is most readily observed for isolated nu- from bootstrap analysis. cleosomes when the overall nucleosome occupancy on DNA is low, but we saw no evidence of nucleosome eviction from the ACKNOWLEDGMENTS. We thank Lorraine Symington (Columbia University) DNA. Given this, the inhibition of end resection observed at for helpful discussions; Hengyao Niu (Indiana University Bloomington) for higher nucleosome occupancies likely arises as the unidirectional Top3 and Rmi1 expression constructs; and members of the E.C.G. and P.S. relocalization of nucleosomes to generate a tightly packed chro- laboratories for comments on the manuscript. This research was funded by matin structure that ultimately becomes prohibitive to Sgs1 NIH Grants R35GM118026 (to E.C.G.), R01ES007061 (to P.S.), CA220123 (to translocation. In this regard, one likely function of chromatin P.S.), and P01CA92584 (to P.S. and E.C.G.), and also the Cancer Prevention & Research Institute of Texas (CPRIT) Recruitment of Established Investigators remodelers is to help remove nucleosomes from dsDNA during (REI) Award RR180029 and P30 CA054174 (to P.S.). J.B.C. is the Mark Foun- Sgs1–Dna2-mediated DNA end resection, so as to alleviate the dation for Cancer Research Fellow for the Damon–Runyon Cancer Research topological impediment stemming from nucleosome mobilization. Foundation Award DRG 2310–17.

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