A new study, conducted by Dr. ZHOU Zheng’s group from the Institute of Biophysics of the Chinese Academy of Sciences and Dr. GONG Zihua’s group from Cleveland Clinic Lerner Research Institute, has revealed the underlying mechanism. This study, published online in Journal of Biological Chemistry, enhances the understanding of how the DSB recruitment module assembles within Shieldin complex.

In this study, after several rounds of screening, Dr. ZHOU’s team firstly identified the minimal REV7-binding domain (RBD) in SHLD3. Through Isothermal Titration Calorimetry (ITC), SHLD3-RBD was shown to have a high-affinity binding ability to REV7 (with low-nanomolar affinity).

The researchers then assembled stable SHLD3-REV7 complex and determined high-resolution complex structures by using X-ray crystallography. The structures revealed that SHLD3-RBD binds REV7 in a unique ladle-shaped conformation with its N-terminal loop and C-terminal α-helix (αC-helix) acting as “stem” and “base”, respectively. Through extensive in vitro and in vivo binding analyses, they found that both parts of SHLD3-RBD are indispensable for REV7 recognition.

In addition, via surface plasmon resonance (SPR) assay, the researchers presented a binding kinetic view of REV7-SHLD3 interaction. The results showed that the “safety-belt” region, which plays a role in binding other proteins, is essential for SHLD3-REV7 binding, as this region retards the dissociation of the RBD from the bound REV7.

This study revealed the molecular basis of the SHLD3-REV7 interaction, provided critical insights into how SHLD3 recruits REV7, and paved the way for medicine development towards cancer treatment.

The staff of the BL-17U1 and BL-19U1 beamlines at the Shanghai Synchrotron Radiation Facility and the Institute of Biophysics have provided technical support to this work.

Overall structure of SHLD3-REV7 complex and their roles in DSB repair (Image by Dr. ZHOU Zheng’s lab)