Structural and Functional Interaction between the Human DNA Repair Proteins DNA Ligase IV and XRCC4

Abstract

Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the highresolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.DNA double-strand breaks (DSBs) represent the most-toxic form of DNA damage in the genome. If left unrepaired, DSBs can result in large-scale loss of genetic information during cell division and, consequently, cell death. DSBs are formed not only in response to endogenous cellular processes, such as V(D)J recombination and oxidative metabolism, but also to various genotoxic agents, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors (38).

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

“…The shorter X4 1-157 construct corresponded to a protein with a central coiled-coil that contained approximately 12 fewer helical turns (46 amino acids) than X4 1-203 construct. X4 1-157 did not contain the X4 residues involved in the L4 interaction (154-188) (21). Calorimetry measurements (Table 1) showed that X4 1-157 interacted with Cernunnos with a dissociation constant (Kd) of 4.1 AE 0.2 μM, which was similar to that obtained previously in studies using the longer construct X4 1-203 (Kd ¼ 3.9 AE 0.2 μM) (29).…”

“…The association between Cernunnos and L4-X4 complex is mediated primarily through an interaction with X4 (14), although a weak interaction with L4 has also been reported (16). Cernunnos stimulates the ligase activity of the L4-X4 complex (20,21,(23)(24)(25) and seems particularly important for the ligation of mismatched or noncohesive DNA ends (25,26). Like X4, Cernunnos is a homodimer (27,28).…”

“…Cells deficient for X4 present no stable L4 (19), whereas L4-deficient cells present a reduced level of X4 (20). Structural studies of the L4-X4 complex revealed a stoichiometry of one L4 to one X4 homodimer and extensive protein-protein interactions mediated by the coiledcoil tails of X4 and the carboxy-terminal tandem BRCT repeat of L4 (21,22). Cernunnos and the L4-X4 complex coprecipitate from crude extract (14, 15) through an interaction sensitive to ionic strength (20).…”

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

“…We also examined the binding pocket in the BRCT domains of Ligase4, XRCC1, BRCA1, and MDC1 because the structure of these BRCT domains has been solved (Zhang et al 1998;Sibanda et al 2001;Shiozaki et al 2004;Stucki et al 2005;Wu et al 2009;Campbell et al 2010;Cuneo et al 2011). However, the binding pockets in the BRCT domains of BRCA1 and MDC1 are much larger compared with those in the FHA domain.…”

Reprint of “The clinical impact of deficiency in DNA non-homologous end-joining”

“…The region between the two BRCT motifs of DNA ligase IV and part of the second BRCT motif are required for binding to XRCC4 [25,26]. DNA ligase IV and XRCC4 interact tightly and regulate one another's stability [27][28][29]. XLF, in contrast, is a weaker binding component of the ligation complex [30].…”