TY - JOUR
T1 - DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks
AU - Yu, Yaping
AU - Mahaney, Brandi L.
AU - Yano, Ken Ichi
AU - Ye, Ruiqiong
AU - Fang, Shujuan
AU - Douglas, Pauline
AU - Chen, David J.
AU - Lees-Miller, Susan P.
N1 - Funding Information:
We thank Dr G. Smith of KuDos Pharmaceuticals Inc. for the generous gift of NU7441 and KU55933, Dr R. Zhang of WEMB for mass spectrometry analysis, S.-Y Wang for her expert help with live cell imaging, Dr P. Jeggo (University of Sussex) for 2BN cells, Dr A. Goodarzi and members of the Jeggo laboratory for providing their transfection conditions for 2BN cells, and D. Boland at SACRI Antibody Services for preparation of antisera. We also thank members of the Lees-Miller laboratory for helpful comments and discussions. This work was supported by grant # 13639 from the Canadian Institutes of Health Research (SPLM) and NIH grants CA50519 and PO1-CA92584 (DJC). SPLM is a Scientist of the Alberta Heritage Foundation for Medical Research and holds the Engineered Air Chair in Cancer Research at the Southern Alberta Cancer Research Institute.
PY - 2008/10/1
Y1 - 2008/10/1
N2 - 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.
AB - 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.
KW - DNA-PK
KW - Nonhomologous end joining
KW - Phosphorylation
KW - XLF
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U2 - 10.1016/j.dnarep.2008.06.015
DO - 10.1016/j.dnarep.2008.06.015
M3 - Article
C2 - 18644470
AN - SCOPUS:52049117396
SN - 1568-7864
VL - 7
SP - 1680
EP - 1692
JO - DNA repair
JF - DNA repair
IS - 10
ER -