30. Oct. 2023
The research groups of Dr. Konstantinos Tripsianes from CEITEC Masaryk University and associated prof. Lumír Krejčí from Masaryk University have expanded the knowledge about the role of the so-called intrinsically disordered region (IDR) of the human RECQ4 protein. The physical and chemical characteristics of the IDR are responsible for diverse RECQ4 functions and its ability to cluster with specific DNA structures named G-quadruplexes. The findings offer interesting perspectives in understanding RECQ4 function in the cell in terms of how proteins are organized into condensates.
At the beginning of its sequence, the RECQ4 protein contains an intrinsically disordered region, meaning that it does not have a defined three-dimensional structure like the rest of the protein. Interestingly, removal of this disordered segment of RECQ4 is lethal in cultured cells. The researchers found that the IDR resembles a Swiss army knife offering RECQ4 the molecular tools to participate in different cellular processes depending on the binding partner. When the RECQ4 IDR binds to replication protein A (RPA), a highly abundant protein capable of binding to single-stranded DNA, it goes from a disordered structure to a static helical structure. When interacting with various DNA structures, it remains unfolded and very dynamic. When the RECQ4 IDR binds with specific DNA structures, called G-quadruplexes (a type of secondary structure of nucleic acids), liquid-liquid phase separation (LLPS) occurs, similar to when droplets of oil appear in a mixture of oil and water.
“These binding modes suggest a regulatory role for the IDR to enable diverse RECQ4 functions in DNA replication and DNA repair in general. Until now, there have been very few studies that have focused on the disordered region of the RECQ4 protein, so our study published in Nature Communications journal broadens the known functional repertoire of IDRs,” says Konstantinos Tripsianes.
Their contribution was a truly cross-disciplinary research effort funded by the Grant Agency of Masaryk University targeting INTERDISCIPLINARY RESEARCH PROJECTS within the University (MUNI/G/1594/2019). To demonstrate and quantify the interactions of the disordered region of the RECQ4 protein, it was necessary to use a combination of biochemical, biophysical, and structural biological methods, including, but not limited to, solution NMR spectroscopy at the Josef Dadok National NMR Center, or optical microscopy, which was the main method used to visualize and characterize LLPS. This integrated approach allowed to bridge microscopic measurements and macroscopic observations into a three-stage kinetic model of LLPS, perhaps one of the first attempts to describe this molecular phenomenon involved in many cellular functions.
Most of the biochemical, biophysical and NMR experiments were carried out by Dr. Anna Papageorgiou, a PhD student in Tripsianes group, while additional biophysical experiments and microscopy to characterize LLPS were performed by Michaela Pospíšilová, a PhD student in Krejčí group. The numerical simulations were performed by Prof. Zbyněk Prokop (MUNI) and Dr. Christopher Waudby (UCL, UK).