Advancing the study of protein-G4 interactions in DNA repair: Insights from biolayer interferometry.

Biolayer interferometry (BLI) is a powerful tool that enables direct observations of protein-G4 interactions in real-time. In this article, we discuss the crucial aspects in conducting a BLI experiment by using the TAR DNA-binding protein (TDP43) and a G4 DNA formed by (GGGGCC)4 as a sample application. We also describe the necessary precautions in designing the DNA substrate and evaluating the signal contributions arising from nonspecific binding interactions. A comprehensive guide is included that details the necessary materials and reagents, experimental procedures, and data analysis methods for researchers who are interested in using BLI for similar studies. The insights provided in this article will allow researchers to harness the potential of BLI and unravel the complexities of protein-G4 interactions with precision and confidence.

Assembly of a G-Quadruplex Repair Complex by the FANCJ DNA Helicase and the REV1 Polymerase.

The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.