binding of doubly functionalized linear dna substrate to streptavidin-coated magnetic beads

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

DNA replication in eukaryotes is carried out by a dynamic protein complex known as the replisome1. A key component of this complex is the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG), which drives and centrally organizes the replisome, leading the way at the front of replication forks1,2. Obtaining a deep quantitative understanding of the dynamics of CMG is therefore critical to understanding the dynamics of the replisome. Such an understanding could be acquired with single-molecule techniques, which are uniquely suited to study molecular motors, such as CMG, due to their unmatched spatial and temporal resolution, and can provide us with an unparalleled quantitative understanding of their function, stochasticity, and dynamics2,3,4,5,6,7,8,9.
In vivo, CMG is loaded and activated in temporally separated fashion to ensure that replication occurs only once per cell cycle1,10,11. First, in the G1 phase of the cell cycle, a set of proteins known as loading factors loads the first component of CMG, the Mcm2-7 hexameric complex, onto dsDNA12,13,14,15,16 in the form of double hexamers with&#160;a head-to-head configuration15,17,18. In the specific case of yeast, this initial process occurs at specific DNA sequences known as origins of replication1. Although Mcm2-7 is the motor core of the replicative helicase, it is by itself unable to unwind DNA19 without the two helicase-activating factors Cdc45 and GINS, which need to be recruited to the loaded Mcm2-7 to give rise to fully active CMG11,19,20,21. The process of helicase activation takes place in the S&#160;phase of the cell cycle and starts with the selective phosphorylation of Mcm2-7 double hexamers by the cell cycle-regulated kinase DDK22,23,24. These phosphorylation events facilitate the recruitment of Cdc45 and GINS to the Mcm2-7 double hexamers10,22,23,24,25,26 by a second set of proteins known as firing factors10,11,26. The binding of Cdc45 and GINS gives rise to two sister CMG helicases, which initially enclose both strands of the parental DNA and are still located in a head-to-head configuration11,27. In the final activation step, the firing factor Mcm10 catalyzes the ATP hydrolysis-dependent extrusion of one DNA strand from each sister CMG11. After strand extrusion, sister CMG helicases bypass and separate from each other by translocating along ssDNA in an ATP hydrolysis-dependent manner11,20,21,28, unwinding DNA by sterically excluding the non-translocation strand29. This entire process has been fully reconstituted in vitro from a minimal set of 36 purified S. cerevisiae polypeptides10,11.
Despite the exquisite in vivo regulation of CMG assembly and activation described above, in vitro reconstituted single-molecule motion studies of CMG2,30,31,32,33,34 have thus far relied on pre-activated CMG purified as a complex from cells20,21, missing all the steps prior to its activation and the bidirectional nature of its motion. This pre-activated CMG approach has been the gold standard in the single-molecule field partly due to the biochemical complexity of the fully reconstituted CMG assembly reaction10,11. This biochemical reaction has been challenging to translate from the bulk biochemical level to the single-molecule level for several reasons. First, to maximize reaction efficiencies, the loading and firing factors needed for CMG assembly and activation are required at concentrations in the range of 10-200 nM10,11,27. These ranges of concentration correspond to the high end of what most single-molecule techniques can tolerate, especially when using fluorescently labeled components35. Finally, CMG has evolved to cruise through thousands of base pairs in a cell36,37,38,39. Therefore, to study its motion at a biologically relevant spatial scale, one requires long DNA substrates (typically of lengths in the order of tens of kilobases)30,31,34,40,41,42. Employing such long DNA substrates poses the additional challenge that the longer the DNA substrate is, the more potential non-specific binding sites for proteins and protein aggregates it has. In the case of CMG, the latter point is particularly important, as several of the loading and firing factors involved in CMG assembly and activation contain intrinsically disordered regions43 and are aggregation-prone.
Here, we report a hybrid ensemble and single-molecule assay that allowed the observation and quantification of the motion of CMG after fully reconstituting its assembly and activation from 36 purified S. cerevisiae polypeptides28. This assay relies on the double functionalization of both ends of a DNA substrate with two orthogonal attachment moieties: desthiobiotin and digoxigenin2 (Figure 1A). The first moiety, desthiobiotin, is used to reversibly bind the DNA substrate to streptavidin-coated magnetic beads44 (Figure 1B). Following this, fluorescently labeled CMG is assembled and activated onto the bead-bound DNA, and a magnetic rack is used to purify and wash the resulting magnetic bead-bound DNA:CMG complexes (Figure 1C). In doing so, the excess protein that would otherwise aggregate on the DNA substrate is removed; this provides virtually aggregation-free DNA:CMG complexes. Intact complexes are then eluted from the magnetic beads by the addition of a molar excess of free biotin, which can outcompete the desthiobiotin-streptavidin interaction (Figure 1D). Individual DNA:CMG complexes are then bound between two optically trapped polystyrene beads coated with anti-digoxigenin antibody (anti-Dig); for this step, the second moiety on the DNA, digoxigenin, is used as it can bind to anti-Dig even in a buffer solution containing an excess of free biotin (Figure 1E). Once the DNA:CMG complex is held in place in the optical trap, the motion of fluorescently labeled CMG is imaged with a confocal scanning laser (Figure 1F). We anticipate that this assay can be easily adapted for the study at the single-molecule level of similarly complex DNA:protein interactions.

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

We study the motion dynamics of different components of the eukaryotic replisome at the single molecule level. In order to obtain a deep quantitative understanding of how cells can duplicate their entire genomes, every cell cycle. In a breakthrough in 2015, eukaryotic DNA replication was fully reconstituted in vitro from purified protein components.And this gave us a lot of control over the system. And since then, it's been used extensively to answer questions about different stages of DNA replication with increasing temporal and spatial resolution. And this was done using complementary approaches such as cryo EM and single molecule biophysics.In the single molecule field, one of the biggest challenges with this system is the number of proteins involved and also the concentrations at which these components are required in order to maximize the efficiency of the overall reaction because this can complicate the single molecule imaging. The protocol described here introduces a hybrid approach in which a protein complex is first assembled in an efficient manner using ensemble biochemistry before then being introduced and studied in a single molecule setting. This avoids the introduction of two high protein concentrations at the single molecule level.We illustrate this approach for studying the behavior of the replicative helicase CMG on DNA at the single molecule level following its assembly via the origin based pathway. But this approach can also be used to study the dynamics of many other types of DNA binding protein complexes.

Binding of Doubly Functionalized Linear DNA Substrate to Streptavidin-Coated Magnetic Beads

Proceed to perform the reaction after functionalizing both ends of the linear DNA substrate. First, take four S-400 spin columns and vortex them for at least 30 seconds to resuspend the resin. Then, centrifuge them for one minute at 735G to remove the storage buffer.Transfer the columns to clean 1.5 milliliter tubes. Immediately, add 100 microliters of the doubly functionalized linear DNA solution to each column. Centrifuge the columns for two minutes at 735G to flow the DNA through the column while retaining the unincorporated nucleotides.After pooling together the flow-through containing the DNA from the four columns, measure the volume with a pipette. Next, vortex M-280 streptavidin-coated magnetic beads for 30 seconds to resuspend them. Transfer four milligrams of the resuspended magnetic beads to a clean 1.5 milliliter tube.Place the tube in a magnetic rack for one minute to collect the beads before removing the storage buffer. Resuspend the beads in one milliliter of buffer A by vortexing for five seconds before incubating the beads at room temperature for five minutes. Collect the beads by placing the tube in a magnetic holder for one minute.After removing buffer A, resuspend the beads in 400 microliters of 2X buffer A by pipetting. Then, add 400 microliters of the functionalized DNA solution to the beads, and mix gently by pipetting. Incubate the bead DNA mixture overnight at four degrees Celsius with end over end rotation.The next day, use the magnetic rack to remove the supernatant before calculating the total amount of DNA bound to the magnetic beads. Then, wash the beads sequentially with buffer B and buffer C.Resuspend the beads in 300 microliters of buffer C and make four 75 microliter aliquots.

binding-doubly-functionalized-linear-dna-substrate-to-streptavidin

Research

JoVE Journal

Biochemistry

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

Ensemble Assembly and Activation of Fluorescently Labeled CMG onto Magnetic Bead-Bound DNA for Single Molecule Imaging

To begin, take one 75 microliter aliquot containing one milligram of DNA-bound streptavidin-coated magnetic beads and remove the supernatant with the magnetic rack. Wash the beads with 200 microliters of loading buffer. Then resuspend the beads in 75 microliters of loading buffer and mix gently by pipetting.Next, add the origin recognition complex at a final concentration of 37.5 nanomolar to the bead-bound DNA and incubate the reaction for five minutes at 30 degrees Celsius with 800 revolutions per minute agitation. Add CDC6 at a final concentration of 50 nanomolar and incubate the reaction as demonstrated before. Then add Mcm2-7 Cdt1 at a final concentration of 100 nanomolar and incubate the reaction for 20 minutes as demonstrated previously.After that, add DDK at a final concentration of 100 nanomolar and incubate similarly for 30 minutes. After removing the supernatant, wash the bead-bound DNA containing phosphorylated Mcm-2-7 hexamers with 200 microliters of high salt wash buffer. Mix by pipetting, ensuring the beads are fully resuspended.Next, remove the buffer and wash the beads once with 200 microliters of CMG buffer. To assemble and activate the fluorescently-labeled CMG onto bead-bound DNA, remove the CMG buffer and resuspend the DNA-bound beads in 50 microliters of CMG buffer supplemented with five millimolar ADP. Next, mix all the components mentioned on the screen in one tube and place it on ice.Immediately add the resuspended bead-bound DNA to the protein mix. Incubate the reaction for 15 minutes at 30 degrees Celsius with 800 RPM agitation. Wash the bead sequentially with HSW buffer and CMG buffer.After removing the buffer, resuspend the CMG containing DNA-bound magnetic beads in 200 microliters of elution buffer, then incubate at room temperature for one hour with 800 RPM agitation. Place the tube in a magnetic rack and allow the beads to be collected for five minutes. Carefully collect the supernatant containing the eluted DNA-CMG complexes without disrupting the settled beads and transfer it to a new tube.To ensure that no beads remain in the solution, place the collected supernatant again in a magnetic rack and keep for another five minutes carefully. Collect the supernatant and transfer it to a new tube. Add 1, 400 microliters of CMG buffer to the 200 microliters of supernatant.The sample is now ready for single molecule imaging.

Single-Molecule Imaging of Fully Reconstituted CMG with Correlative Dual-Beam Optical Tweezers and Confocal Microscopy

Perform the single molecule imaging in a commercial setup, combining dual beam optical tweezers with confocal microscopy. Use inject channel one for bead trapping, channel two for DNA protein complex binding, channel three for checking the presence of CMG and channels four and five as protein reservoirs and buffer exchange locations. Flow all solutions into the flow cell at a constant pressure of 0.5 bar in the injection port.Then turn off the flow in channels four and five. After initially flowing all solutions, reduce the pressure to 0.2 bar. Move trapping lasers to channel one until one bead is caught in each optical trap.Move trapped beads to channel two by moving the joystick while pressing the trigger. Using the joystick without pressing the trigger fish a DNA CMG complex by moving the right bead towards and away from the left bead until a DNA tether is trapped. Move the beads to channel three and immediately stop the flow in all channels.Adjust the 561 nanometer laser power to four microwatts in the excitation laser panel and then take a one frame test scan of the DNA in channel three. If present, CMG will appear as two dimensional diffraction limited spots. In this case, move the DNA tether to either channel four or five for imaging.Otherwise, turn the flow back on, discard the beads and repeat the steps. In channels four or five input two piconewtons in the force spectroscopy panel, and click on the enable button to start a force clamp. Once the initial tension reaches two piconewtons, disable the force clamp before imaging by clicking again on the enable button in the force spectroscopy panel.Click the start scan button in the image scan panel. Image fluorescent CMG every five seconds with a 561 nanometer laser at a power of four microwatts as measured at the objective. In an aggregation free reaction, CMG appeared as discrete, symmetrical diffraction limited spots sparsely crowding the DNA.On the contrary, aggregates are less discrete, sometimes asymmetrical blobs crowding a larger length of the DNA. If the assay was successfully executed and high purity of the purified proteins was achieved, long range motion of CMG in the presence of ATP could be observed.