APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks

Iles N, Rulten S, El-Khamisy SF, Caldecott KW
Source: Mol Cell Biol
Publication Date: (2007)
Issue: 27(10): 3793-803
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Species: human
Tissue Origin: brain
Species: human
Tissue Origin: lung
Nucleofector™ I/II/2b
To knock down APLF in SH-SY5Y cells, 2 x 10^6 cells were cotransfected with pCD2E vector (2 µg) and either pmax-GFP vector (4 µg; Amaxa), pSUPER vector harboring the APLF RNAi sequence GAAGAAATCTGCAAAGATA (4 µg), or 160 pg of the XRCC1 small interfering RNA (siRNA) duplex GCCUGAAGUAUGUGCUA UAdTdT (sense strand; QIAGEN) by using Nucleofector and Nucleofector kit V (Amaxa). Twenty-four hours after nucleofection, cells were placed into a mild selection/differentiation medium containing 0.25 mg/ml G418 and 10 µM alltrans retinoic acid to induce neuronal differentiation. For gamma-H2AX assays, A549 cells stably transfected with pCD2E and either empty pSUPER or pSUPER encoding the APLF RNAi sequence GAAGAAATCTGCAAAGATA, or SH-SY5Y cells 6 days after transient transfection with green fluorescent protein, pSUPER-APLF, or XRCC1 siRNA.
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.