4.6 Effects of IHF binding on DNA minicircles
4.7.2 The role of IHF binding multi-modality
IHF, as with many NAPs has many binding modes as it performs a wide variety of functions in the cell. Both bridging and bending was observed, with the bending occuring in at least three modes.
The multiple (semi-)stable binding modes could be to allow flexibility around an IHF-bound complex, allowing the DNA to partially unwind (so that e.g. other proteins in a transcription complex can bind) without the IHF falling off the DNA completely. A transition between IHF binding states has been seen under tension [120], again
shows the role of tension along the DNA in modulating nucleoid-associated protein (NAP) behaviour. The two non-specific binding modes would also allow for IHF to compact DNA when at high concentration as well as allowing IHF to bridge DNA.
The clustering of DNA observed in the presence of IHF lends support to its role in stabilising extracellular DNA in biofilms or organising the nucleoid. Although this has been seen before (and also shown above) at high concentrations, the DNA constructs with three binding sites also show large cluster formation at small concentrations, from as low as five times that of the DNA. Although extracellular DNA is usually tens of kilobases long [216], it has been seen as short at 400 bp [217]. More importantly the selection of what DNA is excreted by the cell, to include regions which are preferentially bound by multiple IHF would provide a basis for formation of an extracellular DNA matrix within a biofilm at realistic IHF concentrations. Wang et al. [121] observed IHF clusters in E. coli, and these could be formed by regions that contain many close IHF binding sites, helping to organise the nucleoid.
To better understand the role of IHF in the nucleoid, DNA minicircles are a promising avenue of investigation. Minicircles are likely to form kinks to relieve torsion [218] and IHF could therefore play a role in minicircles in the same manner as the nucleoid, while still allowing for single-molecule measurements such as AFM. The preliminary work shown in Section 4.6 suggests there may be a similar multi- modal behaviour although the angles may shift as the DNA is no longer linear. The data also appear to show that compaction is the primary outcome of IHF binding but higher resolution data may allow for the DNA contour to be more accurately determined allowing for a better determination of bending angles.
Minicircles could be studied further with TPM, but in this case tethered-fluorophore motion may be more appropriate (see Section 3.8.3). As circular DNA coils it has a smaller radius of gyration (so the effect of even a 20 nm bead would be larger). This can be seen in Figure 4.14(a), where the minicircles have a radius of approximately 20 nm. However, TFM does have the advantage of being able to use multiple fluo- rophores with multi-colour imaging. This would allow FRET to be simultaneously carried out to determine, for example, the distance around an IHF binding site.
C
H A P T E5
BUILDING AN
AXIAL
OPTICAL
TRAP
T
o better resolve the behaviour of nucleoid-associated proteins (NAPs) one needs to be able to investigate their behaviour on short strands of DNA because they can bind non-specifically and sometimes make small changes on DNA that are not detectable if the DNA is too long. Therefore, in this chapter I describe my building upon previous work in the field in designing optical tweezers that can apply force - in a well-calibrated manner - in the axial direction. I success- fully stretched DNA axially and worked towards building an all-optical/passive force clamp by accurately measuring the optical potential axially, to find the region of constant force.5.1
Why axial tweezers?
Optical tweezers are a fantastic tool to study the interactions of DNA and polymers but are limited by the length of the DNA tether. Efficient trapping of microspheres require the diameter to be around the wavelength of the trapping laser, which is usually an NIR laser (Nd:YAG, 1064 nm). This sets a lower limit on the length of tether for lateral optical tweezers (Fig. 5.1(a)) before the bead is moved so far down that its motion in the axial direction pulls it out of the trap. For a dumbbell trap the interference of two traps (or an oscillation of a time-shared dumbbell trap) prevent accurate measurement (Fig. 5.1(b)). However, moving the trap in the axial direction
(a) (b) (c)
FIGURE5.1. Three examples of possible trapping geometries with optical
tweezers. The dashed arrow shows the movement of the trap and the blue arrow the movement of the trapped microsphere(s). (a) A dumbbell trap uses two beams to hold a tether. This requires a more complicated optical setup but holds a tether in the plane and can be used to achieve base pair resolution more easily than other methods. It also provides a convenient approach to image the tether itself, particularly with fluorescence imaging. However, interference between the two beams sets a lower limit on the tether length. (b) Many optical traps apply force by moving the laser laterally. However, this can cause the bead to be pulled downwards at extreme lengths, and is particularly challenging for short tethers which are comparable to the size of the bead. (c) If instead a single trap moves axially the tether is not held at an angle and so the tether can in theory be arbitrarily short. However, surface effects, a weaker trap in the z direction and more difficult detection can complicate this measurement so it is rarely used.
ensures the motion of the bead is on the same axis as the trap movement (Fig. 5.1(c)) allowing a tether to be much shorter [219].