DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks.
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 mu and lambda 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.
Yu
Yaping
Y
Department of Biochemistry and Molecular Biology, and The Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada.
Mahaney
Brandi L
BL
Yano
Ken-Ichi
K
Ye
Ruiqiong
R
Fang
Shujuan
S
Douglas
Pauline
P
Chen
David J
DJ
Lees-Miller
Susan P
SP
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002470:Cell Survival / Q000528:radiation effects; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000528:radiation effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000528:radiation effects; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D000596:Amino Acids; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050505:Mutant Proteins; C508551:NHEJ1 protein, human; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D010768:Phosphoserine; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D045643:DNA Repair Enzymes