The main objective of this project was to determine the molecular mechanisms that unwind distinct G4s during replication.
The first step in the project was to generate ssDNA templates with distinct G4s at a defined location in the plasmid. Thus, I used my knowledge in structural biology and biophysics to design G4 sequences capable to fold into different G-quadruplex conformations (e.g. different number of G-rich tracks or different loop sequences). These sequences were then cloned into double-stranded plasmids and ssDNA templates generated from these plasmids using M130K7 bacteriophages.
Once I was capable to generate these G4-containing ssDNA plasmids in high concentrations, I replicated these templates in the Xenopus egg extract to determine if distinct G4s are unwound with different kinetics. Thus, initial results showed that different G4 conformations are unwound at different speeds in extract during DNA replication. These results suggest that distinct G4s are unwound with different mechanisms in our system.
Encouraged by the possibility of finding novel proteins involved in regulating different G4 conformations, I setup a DNA pull down assay in combination with mass spectrometry. With this system, when G4-containing templates were replicated in the Xenopus egg extract and the stalling at the G4 was maximum, I stopped the reaction and isolated the DNA and the proteins bound to it. These proteins were then identified by mass spectrometry. By comparing the proteins bound to the control (non-G4 templates) to the ones bound to the G4-plasmids I could determine numerous proteins that bind selectively to G4 DNA. Three specific candidates were selected based on protein enrichment (compared with non-G4s) and protein functionality. We then generated antibodies against these proteins and currently their biochemical functionality in G4 unwinding in the Xenopus egg extract is being tested. Once these validations are successful, these results will be of high interest to the G4 field and will be published in peer-reviewed journals.
Overview of the results
The main conclusions of this action are:
- Distinct G4 structures show different stabilities and unwinding kinetics in the Xenopus egg extract.
- Specific factors that bind selectively to G4 DNA have been identified by mass spectrometry. We ordered antibodies against three newly identified proteins and validation of their biochemical function in Xenopus egg extract is currently being investigated.
Exploitation and dissemination of results
During this project dissemination of results was taken very seriously and the researcher presented her work in several conferences and meetings.
- Poster in the International Meetings on Quadruplex Nucleic Acids (G4thering 2017), May-June 2017, Prague, Czech Republic.
- Poster in CHAINS (Chemistry as Innovative Science), December 2017, Veldhoven, the Netherlands.
- Hubrecht Institute lunch meeting, May 2017, Utrecht, the Netherlands.
From these conferences and meetings two collaborations with laboratories in the G-quadruplex field were stablished and developed by the researcher.
