Biology subjects

Carney, S. M.

Publications and source records attributed to Carney, S. M..

2 recordsLinked to original sources

bioRxiv2022

Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining

Nonhomologous end joining (NHEJ) is the primary pathway of vertebrate DNA double-strand-break repair. NHEJ polymerases and nucleases can modify DNA ends to render them compatible for ligation, but these enzymes are usually deployed only when necessary for repair of damaged DNA ends, thereby minimizing mutagenesis. Using frog egg extracts, we reveal a structural role for the NHEJ-specific DNA Ligase IV (Lig4) in promoting NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the short-range synaptic complex, in which DNA ends are closely aligned prior to ligation. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of short-range synapsis. In this way, compatible ends can be rapidly ligated without polymerase or nuclease activity, which we previously showed is restricted to the short-range synaptic complex. Our results provide a molecular basis for the fidelity of NHEJ.

biophysics↗

bioRxiv2020

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

biochemistry↗