Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts

Abstract

Non-homologous end joining (NHEJ) repairs the majority of DNA double-strand breaks in human cells, yet the detailed order of events in this process has remained obscure. Here, we describe how to employ Xenopus laevis egg extract for the study of NHEJ. The egg extract is easy to prepare in large quantities, and it performs efficient end joining that requires the core end joining proteins Ku, DNA-PKcs, XLF, XRCC4, and DNA ligase IV. These factors, along with the rest of the soluble proteome, are present at endogenous concentrations, allowing mechanistic analysis in a system that begins to approximate the complexity of cellular end joining. We describe an ensemble assay that monitors covalent joining of DNA ends and fluorescence assays that detect joining of single pairs of DNA ends. The latter assay discerns at least two discrete intermediates in the bridging of DNA ends.

Keywords: DNA double-strand break repair; Förster resonance energy transfer; Nonhomologous end joining; Single-molecule fluorescence; Xenopus egg extract.

Fig. 1

Ensemble end joining assay. (A) Time course of an end joining reaction, showing conversion of linear substrate (lin) into open circular (oc), supercoiled closed-circular (scc), dimeric (di), and multimeric products. Supercoiling of closed-circular DNA in extract arises from nucleosome assembly. Closed-circular (cc) topoisomers are visible between the oc and scc bands. The substrate DNA band is slightly overexposed at the 0-min timepoint. (B) Carrier DNA dependence of end joining. A very low concentration of substrate DNA (~0.05ng/μL) was incubated with an extract containing different amounts of closed-circular carrier DNA. End joining does not occur at all if the overall DNA concentration is too low.

Fig. 2

Schematic of intramolecular and intermolecular single-molecule DNA bridging assays. The intermolecular assay (upper panel) relies on a 100-bp DNA substrate labeled near one end with Cy3 (cyan circle) and tethered to the coverslip at the other end by a biotin–streptavidin attachment (black circle). This is incubated with egg extract containing a second 100-bp DNA labeled near both ends with Cy5 (red circles). Formation of the long-range synaptic complex (Graham et al., 2016) is detected in the inter-molecular assay based on the appearance of a discrete Cy5 spot that colocalizes with a Cy3-labeled DNA on the surface. Subsequent formation of the short-range synaptic complex is indicated by the appearance of FRET between Cy3 and Cy5. The intramolecular DNA substrate (lower panel) consists of a single, 2-kb DNA labeled near one end with Cy3 and near the other end with Cy5 and tethered to a streptavidin-coated coverslip via an internal biotin. Formation of the short-range synaptic complex (Graham et al., 2016) is indicated by appearance of FRET between Cy3 and Cy5.

Fig. 3

Preparation of substrates for intermolecular single-molecule assay.

Fig. 4

Intramolecular FRET substrate preparation.

Fig. 5

Schematic of flowcell assembly (left) and completed flowcell (right).

Fig. 6

Schematic of home-built dual view for separating Cy3 and Cy5 emission signals.

Fig. 7

Example single-molecule traces from the intermolecular (A) and intramolecular (B) NHEJ assays. The upper panel in (A) and (B) shows Cy5 emission with direct excitation by 641nm light. The middle panel shows Cy3 and Cy5 emission with excitation of Cy3 by 532nm light. The lower panel shows calculated FRET efficiency. Substrate schematics are shown to the left. Note that 0s corresponds to the first frame in the particular field of view being imaged, not the time of extract addition. (A) In the intermolecular assay, binding of a Cy5-DNA to a Cy3-DNA on the surface is detected by appearance of Cy5 signal with 641nm excitation (first dashed gray line). Formation of the short-range synaptic complex is indicated by the appearance of a high-FRET signal (second dashed gray line) after a time delay (“t_lag”). (B) A sample trace from the intramolecular assay showing three rounds of short-range complex formation and dissolution, as indicated by increases and decreases in FRET. This trace was acquired in extract immunodepleted of XRCC4-LIG4 and supplemented with catalytically inactive XRCC4-LIG4^K278R complex, which supports short-range complex formation but not ligation.