Poly(ADP-ribosyl)ation by poly(ADP-ribose) polymerases regulates the interaction of many DNA damage and repair factors with sites of DNA strand lesions. The interaction of these factors with poly (ADP-ribose) (PAR) is mediated by specific domains, including the recently identified PAR-binding zinc finger (PBZ) domain. However, the mechanism governing these interactions is unclear. To better understand the PBZ-PAR interaction, we performed a detailed examination of the representative PBZ-containing protein involved in the DNA damage response, aprataxin polynucleotide-kinase-like factor (APLF), which possesses two tandem PBZ domains. Here we present structural and biochemical studies that identify Y381/Y386 and Y423/Y428 residues in the conserved C(M/P)Y and CYR motifs within each APLF PBZ domain that are critical for the interaction with the adenine ring of ADP-ribose. Basic residues (R387 and R429 in the first and second PBZ domains, respectively) coordinate additional interactions with the phosphate backbone of ADP-ribose, suggesting that APLF binds to multiple ADP-ribose residues along PAR polymers. These C(M/P)Y and CYR motifs form a basic/hydrophobic pocket within a variant zinc finger structure and are required for APLF recruitment to sites of DNA damage in vivo.DNA damage signaling | high affinity T he DNA damage response (DDR) and maintenance of chromosomal stability is regulated in part by posttranslational modifications, including poly(ADP-ribosyl)ation by poly(ADPribose) polymerases (PARPs), which direct the recruitment of proteins involved in the signaling and repair of DNA damage. In addition, PARP enzymes are important regulators of chromatin remodeling, apoptosis, and transcription (1). In the early response to DNA damage, PARP1 is the predominant PARP activated by DNA strand lesions and catalyzes the attachment of multiple ADP-ribose (ADPr) units from nicotinamide adenine dinucleotide (NAD þ ) onto target proteins, including PARP1 itself (2). Poly(ADP-ribose) (PAR) accumulated at DNA breaks is subsequently metabolized to ADPr by PAR glycohydrolase (3). To date, PAR-binding motifs have been described in some macrodomains, which also bind to ADPr (4, 5), in a basic residue-rich motif interspersed with hydrophobic amino acids (6), and in the more recently described PAR-binding zinc finger (PBZ) domains (7). PBZ domains, present in proteins either as single or two tandem motifs, are limited to multicellular eukaryotes, and the majority of PBZ-containing proteins have putative roles in PAR metabolism, DNA repair, or DNA damage signaling (7-9).APLF (aprataxin polynucleotide kinase (PNK)-like factor, also known as PALF and Xip1) is a newly identified protein involved in the DDR possessing a forkhead-associated (FHA) domain and two tandem PBZ motifs (7-11) (Fig. 1A). APLF was originally identified based on the similarity of its FHA domain to those of PNK and aprataxin (12), which share functional similarities and direct FHA-and phosphothreonine-dependent interactions with the DNA repair proteins XRCC1 and...

APLF is a forkhead associated-containing protein with poly(ADP-ribose)-binding zinc finger (PBZ) domains, which undergoes ionizing radiation (IR)-induced and Ataxia-Telangiectasia Mutated (ATM)-dependent phosphorylation at serine-116 (Ser116). Here, we demonstrate that the phosphorylation of APLF at Ser116 in human U2OS cells by ATM is dependent on poly(ADP-ribose) polymerase 3 (PARP3) levels and the APLF PBZ domains. The interaction of APLF at sites of DNA damage was diminished by the single substitution of APLF Ser116 to alanine, and the cellular depletion or chemical inhibition of ATM or PARP3 also altered the level of accumulation of APLF at sites of laser-induced DNA damage and impaired the accumulation of Ser116-phosphorylated APLF at IR-induced γH2AX foci in human cells. The data further suggest that ATM and PARP3 participate in a common signalling pathway to facilitate APLF-Ser116 phosphorylation, which, in turn, appears to be required for efficient DNA double-strand break repair kinetics and cell survival following IR. Collectively, these findings provide a more detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that Ser116-APLF phosphorylation facilitates APLF-dependent double-strand break repair.

Background: APLF interacts with Ku and facilitates nonhomologous end joining (NHEJ).Results: APLF possesses a Ku-binding motif, and disruption of the APLF-Ku interaction impairs both NHEJ and the nuclear retention of APLF. Conclusion: The APLF-Ku interaction is functionally important in DNA repair and may be important for APLF stability. Significance: The APLF Ku-binding motif appears to represent a general Ku-binding motif.