Deciphering molecular mechanisms that resolve mutagenic DNA G-quadruplex structures

Project Information

ReXeG

Grant agreement ID: 750035

DOI

10.3030/750035

Project terminated on 15 December 2018

EC signature date 23 February 2017

Start date 1 March 2017

End date 28 February 2019

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 165 598,80

EU contribution

€ 165 598,80

Coordinated by

KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN - KNAW
Netherlands

Periodic Reporting for period 1 - ReXeG (Deciphering molecular mechanisms that resolve mutagenic DNA G-quadruplex structures)

Reporting period: 2017-03-01 to 2019-02-28

Summary of the context and overall objectives of the project

The genetic information encoded in our genome is mainly stored in double helical form in our DNA. However, increasing biological interest has focused the attention on other nucleic acid conformations such as G-quadruplexes (G4). G4s are stable nucleic acids assemblies that can form in guanine-rich sequences and regulate crucial cellular processes such as replication or transcription. Importantly, these stable structures come also at a cost as they are able to induce DNA damage and provoke genomic instability. This DNA damage induction potential is especially relevant and has increased the interest of using G4s as potential therapeutic targets. Using G4 stabilizing drugs has been shown to affect gene expression and kill cancer cells. However, the development of drugs that target G4 structures has been obstructed by the lack of knowledge on G4s regulation in vivo.
Currently, very little is known about the molecular mechanisms that form and resolve G4 structures. Several helicases such as FANCJ, WRN, BLM, Pif1 and DNA2 have been described to unwind these stable assemblies in vitro, however, whether and how they resolve G4s in vivo is poorly understood.
In this project, my overall objective was to decipher the molecular mechanisms that unwind G4s using a unique assay developed in the host laboratory. This assay is based on using the Xenopus egg extract system. This uses DNA replication stalling and bypass at defined G4 structures as a direct readout for G4 stability and unwinding under physiological conditions. This is the only system that supports vertebrate DNA replication outside a cell which has generated groundbreaking discoveries in the mechanism of DNA replication and repair. To decipher the molecular details of G4s unwinding, single-stranded plasmids with preformed G4 structures at specific locations are incubated in the extract and DNA replication starts. Upon stalling at the G4, DNA replication is blocked transiently at the G4 after which the G4 is efficiently unwound and the sequence replicated. Therefore, this system enables us to follow the kinetics of G4 unwinding during DNA replication and to determine which proteins are recruited during this process.

Conclusions of the action

From this work the main achievement has been setting up a biochemical assay that combines the Xenopus egg extract and mass spectrometry to identify specific G4 regulatory factors. Thus, I was able to identify several proteins that selectively bind to G4s during DNA replication. Currently, the biochemical function of these proteins in G4 regulation is being validated in Xenopus egg extract in the host laboratory.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In the last years, the importance of G4 structures in cell regulation has become undeniable. G4 structures are necessary to control crucial biological processes such as DNA replication, transcription, translation and telomere maintenance. However, knowledge on the molecular mechanisms that regulate these structures in vivo is still lagging behind.
In this project, I used a Xenopus egg extract system to decipher the machinery that resolves G4 structures during DNA replication. Using this assay in combination with mass spectrometry, I have identified 3 new proteins that bind selectively to G4 DNA and seem to be involved in G4 regulation. If the biochemical function of these novel factors is validated (currently being investigated in the laboratory) these results have a broad interest for several areas of research such as cell biology and drug development. These results will greatly improve our knowledge in cellular regulation, DNA repair associated mechanisms and protein recruitment in the cell. Moreover, understanding how these proteins recognize and resolve G4s will fuel research to determine the molecular details of protein-protein and protein-DNA interactions. Specific protein-protein and protein-G4 interactions in this machinery are promising target for cancer therapy. G4 stabilization (inefficient G4 unwinding) has been described to kill cancer cells. Therefore, developing drugs that target the machinery that unwinds G4s in vivo is an unexplored potential therapy against cancer.

G4 unwinding during DNA replication