In vitro binding kinetics of DNA double strand break repair proteins Ku70/80 and DNA-PKcs quantified by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Abstract

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells. There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls. Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp, 33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation constant (K d ) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80. Finally, using FCCS it was possible to estimate the apparent K d for DNA-PKcs binding to the DNA-Ku70/80 complex and the induced dissociation of DNA-PKcs from that complex by phosphorylation was observed in real time.