Series Of Toroids Research Articles

Protamine folds DNA into flowers and loop stacks

DNA in sperm undergoes an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is dramatically reorganized in minutes by protamine proteins. Protamines are positively charged proteins that coat negatively charged DNA and fold it into a series of toroids. The exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is to study toroid formation by starting at the “bottom” with folding of short lengths of DNA that form loops and working “up” to more folded structures that occur on longer length scales. We previously measured folding of 200–300 bp of DNA into a loop. Here, we look at folding of intermediate DNA lengths (L = 639–3003 bp) that are 2–10 loops long. We observe two folded structures besides loops that we hypothesize are early intermediates in the toroid formation pathway. At low protamine concentrations (∼0.2 μM), we see that the DNA folds into flowers (structures with multiple loops that are positioned so they look like the petals of a flower). Folding at these concentrations condenses the DNA to 25% of its original length, takes seconds, and is made up of many small bending steps. At higher protamine concentrations (≥2 μM), we observe a second folded structure—the loop stack—where loops are stacked vertically one on top of another. These results lead us to propose a two-step process for folding at this length scale: 1) protamine binds to DNA, bending it into loops and flowers, and 2) flowers collapse into loop stacks. These results highlight how protamine uses a bind-and-bend mechanism to rapidly fold DNA, which may be why protamine can fold the entire sperm genome in minutes.

DNA folding by protamine from loops to stacks.

In sperm, DNA undergoes a dramatic reorganization to a semi-crystalline state. This incredible compaction is the work of positively charged protamine proteins which bend and loop negatively charged DNA into a series of toroids. Interestingly, other positively charged molecules such as spermine or cations such as cobalt (+3) have been shown to form these toroid structures in vitro, indicating that the physics that drives this process is likely to be electrostatic. Still, the exact mechanism and the states in the folding pathway are unknown. We have previously shown that multiple protamine molecules “bind and bend” the DNA into a loop. But, what happens after loop formation? To answer this question, we measured folding by protamine in vitro on DNA molecules (length = 600-3000 bp) that are known to form several loops. Using atomic force microscopy (AFM), we were able to identify several intermediate folded structures: molecules with a single loop (a loop), molecules with two or more loops that were connected at a single location like a flower (flowers), and molecules with two or more loops stacked on top of one another (stacks). Using a tethered particle motion (TPM) assay we measured real-time folding of the DNA into these structures. At low protamine concentrations (0.1 µM), we observed many, long-lived, discrete folded states indicative of protamine bending, but at higher concentrations (>0.1 µM) these states appeared as a single, initial folding event. We hypothesize that this single, initial folding event is protamine bending the DNA all along its length, causing multiple loops. These loops then collapse into a stack. More work is necessary to determine how these stacks eventually form toroids. This work is important for spermiogenesis and DNA condensation, but also may useful in DNA nanoengineering.

Protamine Loops DNA in Multiple Steps

DNA in sperm cells is incredibly compact. This compaction is mediated by the protein protamine, which loops DNA into a series of toroids. We are interested in the first step in the in vitro pathway which involves folding DNA into a loop. Previously, it was thought that DNA loop formation would occur in one-step with a mechanism similar to bacterial transcription factors. Bacterial transcription factors loop DNA by i) binding to one location, ii) waiting for a spontaneous fluctuation in the DNA to cause a loop, and iii) stabilizing the loop by binding to a second location. However, unlike bacterial transcription factors that bind one at a time, protamine is known to coat the DNA, binding every 10 bp or so. Thus, the mechanism of protamine might be different from bacterial transcription factors. To study the mechanism of loop formation, we used a tethered particle motion (TPM) assay to measure the dynamic, real-time looping of single DNA molecules. We observed that folding did not occur in a single step as predicted. Instead, the DNA folded multiple times into long-lived (∼100 s), reversible, folded states. We used an atomic force microscopy (AFM) assay to image the folded structures directly. We observed that the partially folded molecules were partially folded loops—c-shapes or s-shapes—that had a radius of curvature of ∼10 nm. Analysis of the contours of the molecules suggest that protamine is bending the DNA rather than increasing the flexibility of the DNA. Thus, our model is that protamine loops DNA in multiple steps, bending it into a curved structure as it binds. This novel pathway for loop formation may be used by other multivalent cations to loop or bend DNA. For more information, see published work in NAR 2020.

Protamin-Induced DNA Looping

DNA is dramatically condensed in sperm cells by protamine proteins. These protamines bind to the major groove of the DNA and loop the DNA into a series of toroids, creating folded DNA that is near crystalline packing levels. One question of interest is how protamine is able to fold the DNA into a single loop. To investigate this question, we performed in vitro tethered particle motion (TPM) assays on short (200 nm) DNA molecules. In these assays, the DNA is biochemically tethered at one end to a glass coverslip and at the other end to a micron-sized polystyrene particle, forming a tethered particle. When we add low concentrations (0.1-0.3 µM) of protamine, the DNA folds, and the range of motion of the tethered particle decreases as the DNA loops. We followed up these dynamic measurements with static atomic force microscopy (AFM) images of DNA molecules in the presence of protamine. We expected our measurements to show a single folding event as the loop forms, but both measurements showed the presence of intermediate states. These results suggest that DNA looping by protamine is more complex than previously thought, with interesting applications for biomaterials research.

Optimizing Tethered Particle Motion to Measure DNA Compaction by Protamine

During mammalian spermiogenesis, there is a remarkable change in the nuclear morphology whereby the chromatin is completely remodeled. Specifically, the histone proteins that wrap the DNA into nucleosomes are removed, and the DNA is coated with a series of small (∼100-amino-acid), arginine-rich protamines that dramatically compact the DNA into a series of toroids. This replacement and compaction 1) reduces the head size of the sperm to enhance their hydrodynamicity, 2) decreases the likelihood of DNA damage, and 3) removes the epigenetic markers passed on through histone modifications. Here, we directly observe the steps of toroid formation and the underlying mechanics. We use a single molecule biophysics assay where we tether a particle (1-µm-diameter bead) to the DNA and to the sample surface. During DNA folding, the end-to-end length of the DNA decreases, decreasing the motion of the particle. This tethered particle motion (TPM) assay is useful because we do not apply any external forces, allowing us to measure reversible folding events. Previously, we saw ∼200 nm changes in length that suggest that the toroid forms by successive loops. Here, we optimized the assay to enhance efficiency and resolution by improving bead monodispersion, protein binding to the surface, and DNA purification.

Investigating the DNA Folding Mechanics of Protamine

In spermatogenesis, an arginine-rich protein called protamine ultimately replaces ∼85 percent of the histones that bind DNA. After replacement, the protamine proteins cause folding of the DNA into a series of toroids, which allow for a high level of DNA compaction. This replacement and compaction 1) reduces the head size of the sperm to enhance their hydrodynamicity, 2) decreases the likelihood of DNA damage, and 3) removes the epigenetic markers passed on through histone modifications. Yet, little is known about the exact mechanism of protamine-induced DNA folding. Here, we measure DNA compaction using a tethered particle motion assay which allows us to measure folding without applying any external forces. In a tethered particle motion assay, a micron-sized bead is tethered to the surface via a DNA molecule. During DNA folding, the end-to-end length of the DNA decreases, decreasing the motion of the bead. When we measured the motion of the bead in the presence of low protamine concentrations (1 μM) we observed discrete changes in the DNA length that occurred in ∼200 nm increments. We interpret these length changes as sequential looping of the DNA into a toroid. Interestingly, the first step in the looping appears to be reversible while subsequent steps are not, possibly due to inter-loop protamine interactions. We then calculate the energetics for the looping and compare our findings to current models. Our results show how protamine is able to fold DNA into a super-compacted state necessary for efficient sperm delivery, which provide novel insights into such limiting case in epigenetics.

Undergraduate Laboratory on DNA Folding using AFM

Atomic Force Microscopy (AFM) is an exciting biophysical technique capable of imaging biological molecules at near atomic resolution, measuring precise force dynamics of protein unfolding, or applying mechanical stress to cells and measuring their response. Yet, despite its ubiquity in biophysical research, AFM has been almost nonexistent in the undergraduate laboratory. Fortunately, with newer AFM systems becoming more affordable and user-friendly, the possibility of AFM in undergraduate courses is now a reality. Our goal is to design an AFM laboratory for students with an introductory background in physics. In this laboratory, students will (1) image the structure of DNA adhered to a mica cover slip in the absence and presence of a condensing agent (protamine), (2) model the DNA as a worm-like chain or a series of toroids, respectively, and (3) use these images to calculate structural parameters of DNA, such as radius of gyration, persistence length, and contour length. This laboratory will expose undergraduates to some of the fundamental AFM techniques that make it a revolutionary tool in biophysical research.