Cohesin SA2 Is A Sequence Independent DNA Binding Protein That .

cohesin SA2 (STAG2) DNA bindinganalyzed. mtSSB protein predominantly bound tothe expected ssDNA region on the gapped DNAsubstrate, while its binding on the nicked DNAsubstrate was random (Parminder Kaur et al.,unpublished data). In summary, these resultsestablished the presence of a ssDNA gap at thedefined location on the linear gapped DNAsubstrate.Next, to study whether or not SA2specifically binds to ssDNA gaps, we directlycompared SA2 binding on non-gapped (withoutnickase treatment) to gapped DNA substrates(Figure 2B&C). AFM imaging showed that on thenon-gapped DNA substrate, SA2 predominantlybound to the DNA ends and its distribution atinternal sites along the linear DNA fragment wasrandom (Figure 2C). This is consistent withposition distributions of SA2 on telomeric andcentromeric DNA substrates (Figure 1D). In starkcontrast, the presence of an ssDNA gap shifted theSA2 binding from the DNA end to a regionconsistent with the location of the ssDNA gap (23%along the length of the DNA, Figure 2C). Analysisof the fractional occupancies of SA2 on DNAdemonstrated that SA2 displayed high bindingspecificities (S 1994 54) for the ssDNA gap. Inaddition, compared to the size of SA2 moleculespositioned outside the gapped regions (1096 117nm3), at the ssDNA gaps SA2 formed largercomplexes with a broader size distribution(1458 232 nm3, Supplementary Figure S1C).single-stranded overhangs at the terminal ends (4 nt3’ overhang on Cen-end DNA, Figure 1D).To further quantify the SA2 bindingspecificity for DNA ends, we applied the analysisbased on the fractional occupancies of SA2 at DNAends (46). SA2 binding specificities for DNA ends(S DNA binding constant for specific sites/DNAbinding constant for nonspecific sites KSP/KNSP)are 2945 ( 77), 2604 ( 68), and 2129 ( 76),respectively, for T270, Cen-end, and Cen-midDNA substrates. In addition, in contrast to SA2alone, DNA-bound SA2 formed higher-orderoligomeric complexes with average AFM volumesof 1025 ( 88) nm3 and 898 ( 63) nm3, respectively,at DNA ends and internal sites (SupplementaryFigure S1C). Based on the calibration curve relatingprotein molecular weights and AFM volumes (44),these AFM volumes correspond to approximatelyfive, and four SA2 molecules, respectively, at theDNA ends and internal sites. In summary, SA2does not specifically bind to centromericsequences, but binds DNA ends with highspecificities that are independent of DNAsequences and short (4 nt) single-strandedoverhangs.SA2 binds to the ssDNA gap with highspecificitiesPreviously, it was established that cohesindeposition and establishment occur in concert withlagging strand-processing (47). ssDNA gaps areintermediate structures on lagging strand duringDNA replication. To directly test whether or notSA2 binds to ssDNA gaps, we used a previouslyestablished method to generate a linear substratecontaining a ssDNA gap (37 nt) flanked by dsDNAarms (Figure 2A). This method was based on thegeneration of four closely-spaced nicks using DNAnickase and subsequent removal of short ssDNAbetween nicked sites using complementary oligos(48,49). After restriction digestion of the circulargapped DNA, the ssDNA gap is at 470 nt (23%)from one end of the DNA (blunt end, Figure 2A andSupplementary Figure S2A). Based on diagnosticrestriction digestion at the gapped region, DNAgapping efficiencies were typically 85 to 95%(Supplementary Figure S2B). To further confirmthe presence of the ssDNA gap, the positiondistribution of mitochondrial single-stranded DNAbinding protein (mtSSB) on this DNA substrate wasSince DNA nicking is the intermediate stepfor generating DNA gaps, we further tested whetheror not SA2 specifically binds to DNA nicks. First,to evaluate if SA2 displays binding specificities forindividual nick sites, we generated a third DNAsubstrate that is a linear DNA substrate (517 bp)containing a single nick site at 37% from one DNAend (50). DNA nicking was confirmed by theobservation of slower mobility of nicked DNA incomparison with its non-nicked counterpart undergel electrophoresis (Supplementary Figure S3A).On the nicked DNA substrate, SA2 displayedpreferential binding to DNA ends (SupplementaryFigure S3A). In stark contrast to what was observedon the gapped DNA substrate, along the nickedDNA substrate SA2 molecules were randomlydistributed at internal sites (Supplementary FigureS3A). Furthermore, on a DNA substrate containingfive nick sites spatially separated from one another,4

cohesin SA2 (STAG2) DNA bindingAFM imaging further established that SA2 did notshow a preference for nicked sites (SupplementaryFigure S3B). In addition, a previous study showedthat the C-terminus of SA2 confers DNA damagesite targeting specificity on SA1 (51). To furtherunderstand SA2 DNA binding, we investigatedwhether SA2 with C-terminal domain deletionretains DNA binding properties. AFM imagingshowed that SA2 1-1051 retains DNA bindingspecificities for DNA ends (S 1687 82) andssDNA gaps (S 1813 79, Supplementary FigureS4). In contrast, AFM imaging showed that SA1also displays high binding specificity for DNA ends(S 2094 38), but not for the 37-nt ssDNA gap(Figure 2C) or nick sites (Supplementary FigureS5). In summary, these results show that SA2displays high binding specificities for ssDNA gaps,but not DNA nicks. SA2 with C-terminal domaindeletion retains binding specificities for DNA endsand ssDNA gaps.(Figure 3B) (60). The three Ni-NTA moieties on thecircular scaffold of the tris-NTA adaptor bind to aHis-tag with subnanomolar affinities (60). AFMimaging revealed that QDs in the presence of onlyBTtris-NTA did not have significant bindingaffinities for DNA. Under the condition used in thisstudy (SA2:QD 4:1), AFM imaging showed thatthe majority (87%) of the SA2-QD conjugatesdisplayed a single SA2 molecule attached toindividual QDs (Figure S6). The addition of Histagged SA2 to the BTtris-NTA-QD reaction led tothe loading of QDs onto DNA, indicating that QDbinding to DNA tightropes was mediated throughSA2. In addition, SA2-QDs retained DNA bindingspecificities toward ssDNA gaps (SupplementaryFigure S7). To monitor SA2 binding on DNA inreal time, QD-labeled SA2 molecules wereintroduced into the flow cell using a syringe pumpafter DNA tightropes were established betweenpoly-L-Lysine treated silica microspheres. Then theflow was stopped, allowing freely diffusing SA2molecules in solution to bind to DNA tightropes(Figure 3C, Movie S1). On all DNA substrates,SA2-QD molecules on DNA were long lived, with 80% of SA2-QD complexes remaining on DNAtightropes after 2 minutes (N 277). The positionsof SA2-QDs were tracked by Gaussian fitting tointensity profiles to obtain the diffusion constant(41,56,57). Importantly, at the same proteinconcentrations (5 nM in the flow cell), the diffusionconstants of SA2 on λ DNA and DNA tightropescontaining either telomeric or centromericsequences are indistinguishable (Figure 3D,Supplementary Table S1). In addition, the alphafactor (diffusive exponent) was calculated todetermine whether SA2 displayed subdiffusivemotion on DNA. An alpha factor of 1 indicates anunbiased random walk and a value less than 1indicates periods of pausing in the random walkprocess (subdiffusion) (61). Recently, we foundthat SA1 shows telomeric sequence dependentsubdiffusive behavior on DNA, manifested by analpha factor significantly smaller than 1 (alphafactor: 0.69 0.03 on telomeric DNA) (41). SA2displayed free 1D diffusion on centromeric DNA(alpha factor 0.96 0.02) and λ DNA (alphafactor 0.93 0.04) tightropes (SupplementaryTable S1). In comparison, the alpha factorsdisplayed by SA2 on telomeric DNA tightropeswere only slightly (p 0.01) lower (0.86 0.03). Insummary, fluorescence imaging of QD-labeledSA2 carries out sequence-independent unbiased1D diffusion on dsDNATarget search through three-dimensionaldiffusion and/or dynamic movements on DNA,such as 1-dimensional (1D) sliding, jumping, andhopping, are essential for proteins to find theirrecognition sites on DNA (52-55). To understandhow proteins dynamically achieve DNA bindingspecificities, we developed a DNA tightrope assaybased on oblique angle total internal reflectionfluorescence microscopy (TIRFM) imaging of QDlabeled proteins on DNA stretched betweenmicron-sized silica beads (41,56-59). DNAtightropes (at an elongation of 90% of the contourlength) are formed between poly-L-Lysine treatedsilica microspheres using hydrodynamic flow(Figure 3A) (57). To generate longer DNAsubstrates with specific sequences that can spanbetween silica microspheres, we ligated linearDNA fragments containing genomic, telomeric, orcentromeric DNA sequences (Figure 1A) (57).Recently, using the DNA tightrope assay, weobserved that QD-labeled SA1 displays slowsubdiffusive events amid fast unbiased 1Ddiffusion in a telomeric sequence dependentmanner (41).To study SA2-DNA binding dynamics, thestreptavidin-coated QD was conjugated to His-SA2using biotinylated multivalent chelator trisnitrilotriacetic acid (BTtris-NTA) as the linker5

cohesin SA2 (STAG2) DNA bindingcompared the diffusion constant and alpha factor ofmobile SA2 on DNA containing ssDNA gaps and λDNA (untreated or nicked) tightropes (Figure 5A).We introduced nicked sites by incubating λ DNAwith Nt.BstNBI nickase. To remove nickase,nicked λ DNA was further purified using phenolchloroform extraction before being introduced intothe flow cell. λ DNA has over 40 Nt.BstNBInickase sites, with spatial separation ranging from13 bp to over 2000 bp. To observe mobile SA2complexes on DNA tightropes, the final SA2-QDconcentration in the flow cell (0.6 nM) was kept thesame across all DNA substrates but lower than thestandard concentration (5 nM, Figure 3 andSupplementary Table S1). On gapped DNAtightropes, SA2 showed a significant (p 0.02)decrease in the diffusion constant and alpha factor(D 0.01 0.003 µm2 s-1 and alphafactor 0.70 0.05) compared to untreated λ(D 0.13 0.03 µm2 s-1 and alpha factor 0.96 0.03)or nicked λ DNA tightropes (D 0.08 0.03 µm2 s-1and alpha factor 0.94 0.04, Figure 5A).Interestingly, on the gapped DNAtightropes, a subpopulation of mobile SA2molecules (N 21 out of 150) alternated betweenmobile and static binding modes (Figure 5B, MovieS2). These apparent static binding events could bedue to SA2 binding or sliding within a narrow rangebelow the resolution of our imaging platform (16nm after Gaussian fitting) (57). The pair-wisedistance between nearest neighbor static SA2binding positions was 0.60 ( 0.19) µm (N 21),which is consistent with the spacing between twoadjacent ssDNA gaps (2.0 kb) on DNA tightropes(Figure 4A). To further compare SA2 DNA bindingdynamics on different DNA substrates, wecalculated a time interval-based diffusion constant(Dint, Supplementary Figure S8) by mobile SA2using a “sliding window” (40-frame, 2 s) MSDanalysis (41). This analysis indicated that distinctfrom the unbiased 1D diffusion mode (Dint: 1.0 X10-2 μm2 s-1) on the centromeric (SupplementaryFigure S8A), telomeric (Supplementary FigureS8B), and λ DNA (Supplementary Figure S8C),mobile SA2 molecules displayed an additionalpopulation with Dint values centered at 1.0 X10-4μm2 s-1 on gapped DNA tightropes (SupplementaryFigure S8D). Furthermore, we used Dint value of 1.0X 10-4 μm2 s-1 as the threshold value to identifyindividual static binding events. This value is basedon the nominal diffusion constant values measuredSA2 on DNA tightropes directly shows that SA2carries out sequence independent 1D diffusion onDNA tightropes containing telomeric, centromeric,or genomic sequences. These results are consistentwith random position distributions of SA2 on bothtelomeric and centromeric DNA substrates shownin AFM images (Figure 1D).SA2 switches between dsDNA and ssDNA gapbinding modesTo study SA2 DNA binding dynamics onDNA tightropes containing gaps, we introducedssDNA gaps after anchoring ligated DNA betweensilica microspheres (Figure 4A). Generation ofssDNA gaps on DNA tightropes was carried out byintroducing the nickase and complementary oligosin the flow cell, followed by heating it at 55ºC, andwashing with high salt buffers to remove nickase,and excess short ss and dsDNA (Figure 2A).Restriction digestion confirmed the presence ofssDNA gaps on DNA tightropes. YOYO1 stainednon-gapped DNA tightropes between silicamicrospheres disappeared after treatment with threerestriction enzymes targeting the sequencesbetween the nickase recognition sites. In contrast,the gapped DNA tightropes stayed intact. Theseobservations confirmed the establishment ofssDNA gaps on DNA tightropes. Compared to SA2on telomeric (46%), centromeric (24%), and nongapped control DNA (39%), on DNA tightropescontaining ssDNA gaps, a significantly (p 10-6)higher percentage of SA2 molecules were static(81%, Figure 4B&C, Supplementary Table S1). Inaddition, the density of SA2 on gapped DNAtightropesincreasedwithhigherSA2concentrations (compare Figure 4B top and bottompanels). To evaluate whether or not the static SA2binding events occurred at the gapped region, wemeasured the distance between nearest neighborSA2-QD pairs. The distribution of this distanceshows three distinct peaks centered at 0.72, 1.23,and 1.87 μm, respectively (Figure 4D), which areconsistent with the expected spacing betweenssDNA gaps on the ligated DNA tightropes (Figure4A). In stark contrast, on DNA tightropescontaining nicks, the spacing between nearestneighborSA2-QDpairswasrandom(Supplementary Figure S3C).To further confirm that DNA bindingdynamics of SA2 on gapped DNA tightropes isdistinctly different from that on nicked DNA, we6

cohesin SA2 (STAG2) DNA bindingas monomers from the solution; the assembly ofhigher-order SA2 complexes on DNA is promotedthrough 1D diffusion and direct interactionsbetween SA2 molecules on DNA.from static protein-QDs on DNA tightropes (41).This analysis indicated that on the gapped DNAtightropes, mobile SA2 molecules displayed asignificantly (p 0.002) higher percentage ( 20%)of time windows (40-frame, 2 s) in the staticbinding mode (Figure 5C) compared to other DNAsubstrates ( 8% for telomeric, centromeric, λ, andnon-gapped control).Taken together, fluorescence imaging ofQD-labeled SA2 establishes that SA2 alternatesbetween two DNA binding modes on gapped DNA– unbiased 1D diffusion on dsDNA (search mode)and stable binding (recognition mode) at ssDNAgaps.Proteins that maintain continuous closecontact with DNA during sliding are unable tocircumnavigate obstacles posed by another proteinon DNA. In contrast, a hopping mechanism inwhich a protein micro-dissociates and re-associateswith DNA within a distance comparable to orgreater than the dimension of DNA-bound proteinscould enable it to transverse these diffusionbarriers. Previously, single-molecules imaging hasrevealed hopping by a DNA repair protein (Mlh1Pms1) and P53 (62,63). We observed instances ofmobile SA2 molecules (N 4 out of 49 collidingSA2 pairs) bypassing another DNA-bound SA2molecules (Supplementary Figure S9B, Movie S3).This bypass frequency is comparable with what wasobserved with Mlh1-Pms1 (62).SA2 forms higher-order oligomeric complexesand can bypass diffusion barriers on DNAIn AFM images, while SA2 alone mainlyexisted as monomers, SA2 formed higher-orderoligomers on DNA (Supplementary Figure S1C).Consistent with these observations using AFM,SA2-QDs with brighter intensities were observed tobreak up into multiple fainter ones (yellow arrows,Supplementary Figure S9A). This observationindicated that the brighter SA2 complexes werehigher-order oligomers. To determine how SA2dynamically forms higher-order oligomericcomplexes on DNA, we analyzed instances wherea mobile SA2 molecule encountered additionalstationary or mobile SA2 molecules. Theoverwhelming majority (92%, N 49) of SA2-SA2interactions on DNA were collisions that did notform complexes. However, there were cases (8%)of initial separate mobile SA2 molecules thatcollided and then diffused in synchronicity withbrighter intensity than individual molecules (whitearrows, Supplementary Figure S9A). The diffusionconstant of larger oligomers of SA2 on DNAtightropes (N 9 complexes on centromeric,telomeric, and gapped DNA) is 0.01 ( 0.02) μm2 s1, which is 10X slower than individual SA2complexes observed in the DNA tightrope assay.For SA2 oligomers, only 7.6% of the time windows(N 3079) shows Dint values less than 1.0 X 10-4 μm2s-1, which is consistent with the alpha factor(0.92 0.04) and suggests that these higher-orderoligomers of SA2 carried out unbiased 1D diffusionwithout significant pausing events. Combined withthe observation that SA2 by itself mainly exists inthe monomeric form (Supplementary Figure S1A),these results imply that SA2 binds directly to DNASA2 binds to DNA intermediate structuresassociated with DNA repair and replicationTo further investigate DNA structures thatSA2 recognizes, we next used a fluorescenceanisotropy assay and compared SA2 binding tossDNA (66, 45, 25 nt) and dsDNA (66, 45, 25, and15 bp) of different lengths (Figure 6A andSupplementary Figure S10). These experimentsshowed that SA2 binds to double- and singlestranded DNA substrates in a length dependentmanner (Figure 6B&C, Supplementary Table S2).There was no detectable SA2 binding for 25 bpDNA, indicating the binding site size of SA2 ondsDNA is larger than 25 bp (Figure 6C).Importantly, for all ds- and ssDNA substratestested, SA2 displays consistently higher bindingaffinities for ssDNA (66, 45, 25 nt) than for dsDNAat the same length (Supplementary Table S2). Inaddition, SA2 DNA binding affinity for telomericsequences (Kd 88.0 1.5 nM) is comparable to thatfor non-telomeric DNA (Kd 76.2 3.9 nM,Supplementary Table S2).Previous studies have demonstrated therole of the cohesin complex in DNA recombinationand re-start of DNA replication after fork stalling(64,65). Therefore, we investigated a series of DNAsubstrates (overhang, flap, fork, and replicationfork) that mimic DNA recombination, repair, andreplication intermediates (Figure 6A&D, and7

cohesin SA2 (STAG2) DNA bindingof the circle and the tail (Figure 8A). Thereplication fork template was created by generatingan ssDNA tail through nick translation using theKlenow fragment over a 398-bp G-less ca

To further quantify the SA2 binding specificity for DNA ends, we applied analysis the based on the fractional occupancies of SA2 at DNA ends (46). SA2 binding specificities for DNA ends (S DNA binding constant for specific sites/DNA binding constant for nonspecific sites K SP/K NSP) are 2945 ( 77), 2604 ( 68), and 2129 ( 76),