Past Event: Oden Institute Seminar

Bridging the gap: Single-molecule studies of DNA double strand break repair

Joseph J. Loparo, Harvard University

Abstract

NHEJ is the primary repair pathway for double-strand breaks in human cells, yet we lack a detailed understanding of how broken DNA ends are brought together, enzymatically processed, and joined. To better understand these questions, we have developed single-molecule assays to visualize NHEJ in Xenopus egg extracts. Egg extract is a powerful system to study NHEJ because it contains the complete soluble proteome and robustly carries out efficient repair in a manner that depends on all of the core NHEJ factors. Using single-molecule colocalization and FRET assays to watch the association of DNA ends in real time, we recently showed that the NHEJ machinery passes through at least two distinct synaptic states (Graham et al. Mol Cell 2016). DNA ends are initially tethered >50 Å apart in a relatively unstable “long-range” complex that depends on the NHEJ factors Ku and DNA-PKcs. DNA-PKcs kinase activity and the NHEJ factors responsible for ligation (XLF, XRCC4, and LIG4) are then required to transition to a stable “short-range” complex in which the ends are brought into close alignment prior to ligation. In this talk I will describe how we are extending these studies to better understand how the NHEJ machinery assembles on DNA ends and how DNA end processing is regulated to maximize the fidelity of repair.