Open AccessC-NHEJ without indels is robust and requires synergistic function of distinct XLF domains
Author(s) -
Ragini Bhargava,
Manbir Sandhu,
Sanychen Muk,
Gabriella Lee,
Nagarajan Vaidehi,
Jeremy M. Stark
Publication year - 2018
Publication title -
nature communications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.559
H-Index - 365
ISSN - 2041-1723
DOI - 10.1038/s41467-018-04867-5
Subject(s) - indel , non homologous end joining , genetics , dna repair protein xrcc4 , dna , biology , mutation , computational biology , gene , homologous recombination , dna repair , single nucleotide polymorphism , dna mismatch repair , genotype
To investigate the fidelity of canonical non-homologous end joining (C-NHEJ), we developed an assay to detect EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (indels). Here we find that such EJ requires several core C-NHEJ factors, including XLF. Using variants of this assay, we find that C-NHEJ is required for EJ events that use 1–2, but not ≥3, nucleotides of terminal microhomology. We also investigated XLF residues required for EJ without indels, finding that one of two binding domains is essential (L115 or C-terminal lysines that bind XRCC4 and KU/DNA, respectively), and that disruption of one of these domains sensitizes XLF to mutations that affect its dimer interface, which we examined with molecular dynamic simulations. Thus, C-NHEJ, including synergistic function of distinct XLF domains, is required for EJ of chromosomal breaks without indels.