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

Yaping Yu, Brandi L. Mahaney, Ken Ichi Yano, Ruiqiong Ye, Shujuan Fang, Pauline Douglas, David J. Chen, Susan P. Lees-Miller

Research output: Contribution to journalArticle

55 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1680-1692
Number of pages13
JournalDNA Repair
Volume7
Issue number10
DOIs
StatePublished - Oct 1 2008

Fingerprint

Ataxia Telangiectasia
Phosphorylation
Double-Stranded DNA Breaks
Repair
DNA
Polynucleotide 5'-Hydroxyl-Kinase
Serine
Joining
DNA Ligases
DNA-Activated Protein Kinase
Catalytic DNA
Radiation Tolerance
DNA-Directed DNA Polymerase
Alanine
Protein Kinases
DNA Damage
Catalytic Domain
Lasers
Cells
Radiation

Keywords

  • DNA-PK
  • Nonhomologous end joining
  • Phosphorylation
  • XLF

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Yu, Y., Mahaney, B. L., Yano, K. I., Ye, R., Fang, S., Douglas, P., ... Lees-Miller, S. P. (2008). DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks. DNA Repair, 7(10), 1680-1692. https://doi.org/10.1016/j.dnarep.2008.06.015

DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks. / Yu, Yaping; Mahaney, Brandi L.; Yano, Ken Ichi; Ye, Ruiqiong; Fang, Shujuan; Douglas, Pauline; Chen, David J.; Lees-Miller, Susan P.

In: DNA Repair, Vol. 7, No. 10, 01.10.2008, p. 1680-1692.

Research output: Contribution to journalArticle

Yu, Y, Mahaney, BL, Yano, KI, Ye, R, Fang, S, Douglas, P, Chen, DJ & Lees-Miller, SP 2008, 'DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks', DNA Repair, vol. 7, no. 10, pp. 1680-1692. https://doi.org/10.1016/j.dnarep.2008.06.015
Yu, Yaping ; Mahaney, Brandi L. ; Yano, Ken Ichi ; Ye, Ruiqiong ; Fang, Shujuan ; Douglas, Pauline ; Chen, David J. ; Lees-Miller, Susan P. / DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks. In: DNA Repair. 2008 ; Vol. 7, No. 10. pp. 1680-1692.
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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.

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