DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks

Abstract

Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases μ and λ and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.

Cdk1-dependent regulation of the Mre11 complex couples DNA repair pathways to cell cycle progression

“…Consistent with this hypothesis, removal of additional residues (serines/ threonines followed by prolines, aspartates, glycines, and glutamates, Fig. S3A) that are targeted in a number of substrates of ATMlike kinases in vivo [38][39][40][41][42] completely abrogated Xrs2 phosphorylation in response to DNA damaging agents (i.e., xrs2-27A, Fig. S3B).…”

“…This result is consistent with the observation that members of the ATM/ATR family of kinases-like many other kinases-are sometimes capable of phosphorylating substrates on residues that do not fit their canonical consensus motif. [39][40][41][42] It is also important to note that Xrs2 has been predicted to be a target of Rad53 kinase, a central effector of the Tel1/Mec1-dependent checkpoint response. 56 Although this prediction remains to be verified, it is possible that a small, but nevertheless significant, fraction of Xrs2 DNA damage-dependent phosphorylation may be catalyzed by Rad53, thereby explaining the involvement of non-consensus Tel1 sites in this phosphoregulatory process.…”

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

“…Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15)(16)(17)(18); however, recent experiments indicate that DNAPKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered DSBs (19)(20)(21)(22)(23)(24)(25). In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26)(27)(28).…”

The Pathways of Double-Strand Break Repair

“…In addition to its catalytic function at the DSB ends, DNA-PKcs may also tether the broken DNA ends to facilitate rejoining (Meek et al, 2004). Although the DNA-PKcs kinase activity catalyzes the phosphorylation of many NHEJ related substrates, the phosphorylation of the DNA-PKcs itself is the only physiologically relevant event identified so far Cruet-Hennequart et al, 2008;Otsuki et al, 2007;Soubeyrand et al, 2006;Yu et al, 2008). DNA-PKcs autophosphorylation appears to be important for DSB repair as DNA-PKcs mutated at key phosphorylation sites (T2609 and S2056 at ABCDE and PQR clusters respectively) is impaired in its function in D-NHEJ (Cui et al, 2005;Meek et al, 2007).…”

“…DNA-PKcs is a serine/threonine kinase with specificity for S/TQ sites (Marshall, 2011) that regulates its activity through autophosphorylation. It targets, RPA2, WRN, Cernunnos/XLF, LigIV, and XRCC4 Cruet-Hennequart et al, 2008;Otsuki et al, 2007;Soubeyrand et al, 2006;Yu et al, 2008). In addition to its catalytic function at the DSB ends, DNA-PKcs may also tether the broken DNA ends to facilitate rejoining (Meek et al, 2004).…”