TOPBP1interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4 TOPBP1 coprecipitates with Rad3 ATR , suggesting that phosphorylation coordinates formation of an active checkpoint complex. In multicellular eukaryotes, DDRs also interface with the apoptotic and senescence pathways to ensure that specific cell types that receive high levels of damage are removed from the cycling population (Wahl and Carr 2001). Failure of DDRs underlies many cancer-prone human genetic diseases, and mutations in DDR proteins are common events in the etiology of sporadic cancers.Many of the DDRs require the activation of the ATRand ATM-dependent DNA structure checkpoint pathways. Activation of the ATR and ATM kinases promotes a cascade of phosphorylation events. Among these are phosphorylation and activation of two downstream kinases Chk1 and Chk2 (Shiloh 2003). The phosphorylation of target proteins by ATM, ATR, Chk1, and Chk2 results in the regulation of transcription, changes in the profiles of protein stability, and changes to the subcellular localization of certain proteins Shiloh 2003;Yao et al. 2003). Several target proteins have been identified, and in some instances, individual phosphorylation events have been ascribed specific functions. For example, phosphorylation of p53 and its E3 ubiquitin ligase Mdm2 influences the stability and function of p53, an important effector of checkpoint signals (Chene 2003). Because checkpoint pathways are vital for the maintenance of genomic stability and the suppression of carcinogenesis, many studies have aimed to understand the molecular mechanisms underpinning checkpoint pathway activation. Two distinct DNA-structure-responsive checkpoint pathways have been characterized.The ATM-dependent checkpoint responds directly to DNA double-strand breaks (DSBs). Activation of ATM requires the presence of the Mre11-Rad50-Xrs2 NBS1 complex (MRX), and studies in budding yeast demonstrate that the MRX complex binds to DNA doublestrand ends at the site of damage and recruits the ATM homolog Tel1 (Nakada et al. 2003). In human cells, Nbs1 is required for ATM-dependent phosphorylation events in response to DNA damage (Girard et al. 2002;Uziel et al. 2003). ATM phosphorylates MRX in response to DNA damage and also targets a histone H2A variant, several checkpoint mediator proteins (including BRCA1, TOPBP1, P53BP1, and MDC1) and activates Chk2 (Shiloh 2003).The ATR-dependent checkpoint responds to a variety of genotoxic insults, including UV-induced dimers and agents that stall DNA replication. ATR forms a stable protein complex with ATRIP, and this subunit is required to bind ATR-ATRIP to single-stranded DNA
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...
Double-strand breaks repaired by homologous recombination (HR) are first resected to form single-stranded DNA, which binds replication protein A (RPA). RPA attracts mediators that load the Rad51 filament to promote strand invasion, the defining feature of HR. How the resection machinery navigates nucleosome-packaged DNA is poorly understood. Here we report that in Schizosaccharomyces pombe a conserved DDB1-CUL4-associated factor (DCAF), Wdr70, is recruited to DSBs as part of the Cullin4-DDB1 ubiquitin ligase (CRL4Wdr70) and stimulates distal H2B lysine 119 mono-ubiquitination (uH2B). Wdr70 deletion, or uH2B loss, results in increased loading of the checkpoint adaptor and resection inhibitor Crb253BP1, decreased Exo1 association and delayed resection. Wdr70 is dispensable for resection upon Crb253BP1 loss, or when the Set9 methyltransferase that creates docking sites for Crb2 is deleted. Finally, we establish that this histone regulatory cascade similarly controls DSB resection in human cells.
The Acknowledgment section was inadvertently omitted and should appear as shown below. ACKNOWLEDGMENTS F.C.-L. is a recipient of an EMBO long-term fellowship (ALTF956-2006). K.W.C. is funded by the MRC (G0400959 and G0600776), BBSRC (BB/C516595/1), and the European Community (integrated project DNA Repair LSHG-CT-2005-512113).
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