The task was to monitor the HIV-Integrase Nucleosome Complex Assembly
As soon as in-plate DLS (SpectroLight 600) comes into action, the insights level up!
Receiving inspection before running the complex assembly experiments. Conclusion: All subunits were ready!
The experimental run began with a buffer screen
Providing a little background information can lead to surprisingly conclusive results, as demonstrated by the following summary.
Overview of the assignment and in plate DLS screening results.
Verification of the remarkable stability of the nucleosome
The complete nucleosome hydrodynamic radius was measured to be ~7.1 nm, even storage for 7 days at 30°C had no effect in terms of size changes of this particle.
Discovery of the free-runnig non-specific endonuclease activity of IN/40bp vDNA
Attempting to assemble a nucleosome-IN/40 bp vDNA complex without the host protein LEDGF was unsuccessful. Mixing nucleosomes with IN/40 bp vDNA resulted in the formation of larger and smaller particles. This progression continued for over a week. Based on this result, a hypothesis could be formulated.
Hypothesis: In the absence of LEDGF, IN does not form specific interactions and therefore acts as a free-running, non-specific endonuclease. The creation of multiple DNA cuts eventually results in nucleosome disintegration. Released histones are poorly soluble and form large aggregates, whereas the DNA of various length fragments are soluble and appear as a much smaller peak. This remains to be verified.
Regarding the question marks, another hypothesis can be proposed: Since the endonuclease active site requires DNA binding, only DNA fragments with a minimum length can be cleaved by IN. Therefore, the fragments became more uniform close to this minimum size. Short DNA fragments can bind to histones and make them water-soluble. This remains to be verified as well.
The fixation of IN/40 bp vDNA to nucleosomes by LEDGF inhibited IN's endonuclease activity
Human lens epithelium-derived growth factor (LEDGF) is known to be recruited by the viral protein IN, which guides IN-attached viral DNA to potential integration sites. This complex remained stable for at least seven days under certain buffer conditions. This observation can be used to formulate another hypothesis.
Hypothesis: HIV-Integrase is fixed by the specific binding of LEDGF to the nucleosome. This fixation inhibits the ability of IN to act as a free-running, non-specific endonuclease. To be verified...
The study was completed by long term stability analysis of the complex subunits
The HIV integrase undergoes a transition to an oligomer or aggregate that has not been described before within five days at 30 °C.
The LEDGF is already polydisperse at the beginning and has a tendency to aggregate further at 30 °C over a period of 5 days.
The complete nucleosomes were monodisperse at the beginning stays in this state for over 7 days at 30°C.
Verifying the above hypothesis requires a combination of methods, including single-particle 3D cryo-EM. We will support our customers in designing experiments to verify or falsify the hypothesis. Some of the results will be published in one of the next newsletters.
To be continued...
We would like to express our gratitude to our contract partner, who kindly agreed to present some of the results and conclusions. The used hardware was a standard SpectroLight 600 in plate DLS system
Your XtalConcepts Team
