Biophysical measurements of cells, microtubules, and DNA with an atomic force microscope

Devenica, Luka Matej; Contee, Clay; Cabrejo, Raysa; Kurek, Matthew; Deveney, Edward; Carter, Ashley R.

doi:10.1119/1.4941048

“…In this setup, spontaneous looping is more likely to occur for DNA contour lengths, L c , that are much greater than the DNA persistence length, L p . The L p of the DNA (50 nm (40,41)) is the length over which the tangent vector to the DNA remains correlated (42) and is essentially the length that the DNA is fairly stiff and straight. If L c is much longer than L p , then the DNA can bend due to thermal fluctuations, creating a spontaneous loop.…”

Section: Resultsmentioning

confidence: 99%

DNA looping by protamine follows a nonuniform spatial distribution

McMillan

¹

,

Kuntz

²

,

Devenica

³

et al. 2021

Preprint

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DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes (SMC) proteins such as condensin. Here, however, we are interested in a different looping method whereby multivalent cations (charge≥+3), such as protamine proteins, neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤ 1 μm) DNA fragments using atomic force microscopy (AFM). We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it overestimated the number of loops that form at the ends of the molecule and failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine’s in vivo role for looping DNA into toroids and the mechanism of DNA condensation by multivalent cations more broadly.SIGNIFICANCEDNA looping is important in a variety of both in vivo functions (e.g. gene regulation) and in vitro applications (e.g. DNA origami). Here, we sought a mechanistic understanding of DNA looping by multivalent cations (≥+3), which condense DNA into loops and toroids. One such multivalent cation is the protein protamine, which condenses DNA in sperm. We investigated the mechanism for loop formation by protamine and found that the experimental data was consistent with an electrostatic multibinding model in which two protamines bind electrostatically to the DNA within a 50-nm region to form a loop. This model is likely general to all multivalent cations and may be helpful in applications involving toroid formation or DNA nanoengineering.

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“…We chose the longest DNA length (1023 nm) for spontaneous looping experiments because looping is more likely to occur for DNA lengths, L C (contour length or length along the DNA contour), that are much greater than the DNA persistence length, L P . The L P of the DNA (50 nm ( 37 , 38 )) is the length over which the tangent vector to the DNA remains correlated ( 39 ) and is essentially the length that the DNA is fairly stiff and straight. If L C is much longer than L P , then the DNA can bend because of thermal fluctuations, creating a spontaneous loop.…”

Section: Methodsmentioning

confidence: 99%

DNA looping by protamine follows a nonuniform spatial distribution

McMillan

¹

,

Kuntz

²

,

Devenica

³

et al. 2021

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes proteins such as condensin. Here, however, we are interested in a different looping method whereby condensing agents (charge Rþ3) such as protamine proteins neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (%1 mm) DNA fragments using atomic force microscopy. We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine's in vivo role for looping DNA into toroids and the mechanism of DNA condensation by condensing agents more broadly.

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“…There was some DNA shrinkage due to drying in air, but most molecules (70%) were still within 20% of the expected contour length. DNA strands previously imaged with this system show heights of 0.55 ± 0.07 nm and have a lateral radius of 3.9 ± 0.6 nm, close to the expected lateral radius of 3 nm (37). We used a scan rate of either 2-4 Hz.…”

Section: Afm Data Collectionmentioning

confidence: 98%