Structure and identification of ADP-ribose recognition motifs of APLF and role in the DNA damage response

Abstract

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...

Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

“…Imposed by PARP1 and PARP2 enzymes in response to DNA breaks in mammalian cells, where it has been most thoroughly studied, PARylation is one of the earliest events in the DDR but is quickly removed by the action of PARG (PAR glycohydrolase) (Gagne et al 2006;Hakme et al 2008;Krishnakumar and Kraus 2010). Through the use of anti-PAR antibodies, PARylation can be detected locally at sites of DNA breaks in mammalian cells , where it promotes recruitment of the DNA break repair factors XRCC1 (El-Khamisy et al 2003;Okano et al 2003) and APLF Kanno et al 2007;Ahel et al 2008;Rulten et al 2008;Eustermann et al 2010;GY Li et al 2010). PARylation at DNA breaks is also required for accrual of the chromatin remodeling factors ALC1 and CHD4 (Ahel et al 2009;Gottschalk et al 2009;Chou et al 2010;Polo et al 2010), the Polycomb histone-modifying complex (Chou et al 2010), and the histone variant macroH2A ).…”

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

“…Since CHFR and APLF are known PAR-binding proteins, which bind PAR via their PBZ motifs (Ahel et al 2008;Li et al 2010;Oberoi et al 2010), we used GST, recombinant CHFR, and APLF PBZ motifs as negative and positive controls, respectively, to screen PAR-binding domains. We examined 19 FHA or BRCT domains and found that two FHA domains (from PNKP and APTX), two BRCT domains (from Ligase4 and XRCC1), and an FHA-BRCT fusion domain (from NBS1) interacted with PAR (Fig.…”

The oligonucleotide/oligosaccharide-binding fold motif is a poly(ADP-ribose)-binding domain that mediates DNA damage response

“…Upon DNA damage induction, poly(ADP ribose) (PAR) polymerases (PARPs), such as PARP1, the founding member of the PARP family, rapidly detect DNA breaks, including single-strand breaks (SSBs) and double-strand breaks (DSBs), and activate PAR synthesis at or adjacent to DNA lesions (7,8). Recent evidence suggests that DNA damage-induced PAR may serve as a docking signal to recruit DDR factors to DNA lesions (7)(8)(9)(10)(11)(12)(13)(14). For example, our recent study showed that the BRCT domains of BARD1 and NBS1 recognize PAR, which facilitates the rapid recruitment of the BRCA1/BARD1 complex and NBS1 to the site of DNA damage (15,16).…”