APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks.
Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.
Iles
Natasha
N
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, Sussex, United Kingdom.
Rulten
Stuart
S
El-Khamisy
Sherif F
SF
Caldecott
Keith W
KW
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002875:Chromosomes / Q000378:metabolism*; D004249:DNA Damage / Q000528:radiation effects; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays; D000076105:X-ray Repair Cross Complementing Protein 1
D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D010750:Phosphoproteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011993:Recombinant Fusion Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C101467:XRCC4 protein, human; D047390:Casein Kinase II; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; C520557:APLF protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase