Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Abstract

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5 0 -OH and 3 0 -phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.

Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4

“…The Lig4/ Dnl4 BRCT domains bind strongly to XRCC4/Lif1 by encircling this coiled coil at a conserved motif near its C-terminal end [7], supporting robust co-purification of Lig4/Dnl4 with XRCC4/ Lif1 [12][13][14]. Finally, XRCC4/Lif1 contains a C-terminal region that is poorly conserved and structurally uncharacterized, but that is nonetheless phosphorylated and required for NHEJ and interaction with FHA domains of two proteins [15,16] (manuscript in preparation). The precise actions of XRCC4/Lif1 in supporting Lig4/Dnl4 are not clear, but it binds DNA [17] and is required for stability of Lig4/Dnl4 [9,18] and its recruitment to DSBs [19].…”

Non-homologous end-joining, a sticky affair

“…To permit both DNA polymerization and ligation, a 3 0 -OH and a 5 0 -phosphate need to be present at the DNA ends. Polynucleotide kinase is recruited to DNA ends by interaction with XRCC4 and provides the activity to both remove 3 0 -phosphates and add 5 0 -phosphates (Koch et al, 2004).…”

Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation

“…The expression, extraction, and purification of GST fusion proteins were performed as previously described (18).…”