APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

Abstract

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADPribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.The rapid repair of chromosomal DNA single-and doublestrand breaks is critical for genome integrity, and defects in this process result in a variety of hereditary genetic diseases (21). Recently, we and others identified the human protein APLF (aka C2orf13, PALF, and XIP1) as a novel component of the DNA single-strand break repair (SSBR) and double-strand break repair (DSBR) machinery (4,14,15,19). The amino terminus of APLF contains a highly conserved forkhead-associated (FHA) domain that mediates interaction with the SSBR and DSBR factors XRCC1 and XRCC4, respectively. In addition, APLF interacts with Ku80 in an FHA domain-independent manner. The C terminus of APLF contains a second highly conserved region that encodes two tandem zinc fingers (designated ZNF1 and ZNF2) and a highly acidic tail. Both the FHA domain and the tandem ZNFs can facilitate, by independent mechanisms, the accumulation of APLF at sites of DNA strand breakage (4,14,15). Whereas the FHA domain facilitates APLF accumulation via interaction with CK2-phosphorylated XRCC1, the mechanism by which the ZNFs achieve this is unclear.The ZNFs in APLF most closely resemble the tandem zinc fingers present in tristetraprolin. Tristetraprolin binds specific mRNA species, with each of two ZNFs targeting a separate 5Ј-UAUU-3Ј subsite located within a larger, AU-rich recognition sequence (5, 18). Although APLF does not bind this mRNA species (unpublished observations), it is possible that the APLF ZNFs might interact with some other type of adenine-rich structure. One such structure that arises during DNA strand break repair is poly(ADP-ribose) (pADPr), a branched nucleic acid-like polymer synthesized rapidly at DNA strand breaks by pADPr po...

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

“…To date, three PAR-binding motifs have been identified in various proteins, including the putative consensus sequence of an eight amino-acid motif 30,31 , the PAR-binding zinc-finger (PBZ) motif 32,33 and the macrodomains [34][35][36] . Despite the universal nature of the poly(ADP-ribosyl)ation of target proteins, and PARP's function in a wide range of cellular activities 21,25 , only a limited number of PAR-binding motifs have been found in several target proteins 20,37 .…”

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts

“…But, a 70% of knockdown of APLF at the protein level failed to reveal any issue with the stability of iPSCs. APLF, along with its interacting partner PARP3, basically promotes NHEJ (Rulten et al, 2008). Even in the absence of Aplf, overexpression of XRCC4 and DNA ligase IV leads to NHEJ repair (Rulten et al, 2008).…”

“…APLF is an important constituent of the non-homologous end joining (NHEJ)-mediated DNA damage repair machinery (Rulten et al, 2008;Grundy et al, 2013), and its downregulation in human cells could sensitize the cells to various DNA-damaging agents (Macrae et al, 2008). In order to test whether Aplf knockdown induces DNA repair defects, the cells were first challenged with actinomycin D at different concentrations and subjected to an apoptosis assay.…”

BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability

“…PARP1 (and the closely related PARP2) is rapidly activated at sites of DNA breaks, where it catalyzes the formation of poly(ADP-ribose) (PAR) polymers both on itself (autoPARylation) and local substrates (Satoh and Lindahl, 1992;Ame et al, 1999;Allinson et al, 2003;Woodhouse and Dianov, 2008). DNA damage associated PARylation, either directly or through the recruitment of proteins such as APLF and the chromatin remodeling enzyme ALC1, induces changes in local chromatin structure that lead to chromatin relaxation and recruitment of other DNA repair proteins (Tulin and Spradling, 2003;Ahel et al, 2008;Rulten et al, 2008;Gottschalk et al, 2009).…”