A New Player in Overcoming Protein Linked DNA Damage Karim Bahmed1, Karin C Nitiss1, John L Nitiss1

UnTTrapping the ends: A new player in overcoming protein linked DNA damage Karim Bahmed1, Karin C Nitiss1, John L Nitiss1

1Molecular Pharmacology Department, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA Cell Research (2010) 20:122-123. doi:10.1038/cr.2010.17; published online 1 February 2010

DNA topoisomerases carry out their have been implicated in removing topoi- remove phosphotyrosyl linked peptides reactions by generating transient co- somerases covalently bound to DNA, from both the 5′ and 3′ ends of DNA valent phosphotyrosine intermediates most importantly nucleases associated [10]. Since the mammalian enzyme with DNA [1]. This reaction mechanism with the Mre11/Rad50/Nbs1 (MRN) apparently lacks the ability to process allows these enzymes that need to break complex. Specific nuclease deficient 5′ phosphotyrosyl linked peptides DNA to do so in a way that does not mutations in either Mre11 or Rad50 [11], other mammalian enzymes might induce genome rearrangements or DNA prevent the processing of Spo11:DNA process 5′ phosphotyrosyl “adducts”. damage responses. However, a variety complexes arising from Spo11. Spo11 Work by Ledesma and colleagues has of agents are able to trap topoisomerases is a type IIB topoisomerase homolog found an enzyme, TTRAP that has this while they are covalently bound to that introduces the DNA double strand property [12]. DNA. These agents include small mol- breaks that initiate meiotic recombina- Ledesma et al. identified TTRAP ecules that are active and important tion [4]. S. cerevisiae sae2 mutants are in a screen that was designed to de- anti-cancer or anti-bacterial agents also deficient in disjoining Spo11 from tect enzymes besides Tdp1 that could [2, 3]. Trapping of topoisomerases DNA, and work in S. pombe has dem- process 3′ phosphotyrosyl adducts. leads to DNA damage that includes onstrated that the Sae2 homolog Ctp1 They screened a mammalian cDNA strand breaks, and protein covalently (CtIP in mammalian cells) can disjoin library in a yeast strain lacking both bound to DNA. While strand breaks trapped Top2 from DNA [5]. Tdp1 and Rad1. Rad1 is a homolog of can be repaired by conventional break In addition to nuclease removal of mammalian XPF, and ERCC1/XPF is a repair pathways such as homologous trapped topoisomerases, specific phos- heterodimer nuclease that participates recombination and non-homologous phodiesterases are also able to process in nucleotide excision repair, and in end-joining, the removal of protein co- trapped topoisomerases by cleavage other DNA repair pathways. Genetic valently bound to DNA is an interesting of the enzyme:DNA phosphotyrosine evidence suggested that ERCC1/XPF and unusual challenge for cells. linkage. The first identified enzyme is a component of another pathway for There are two major classes of that could carry out this reaction was processing Top1 damage; therefore the enzymatic activities that can repair identified in yeast and termed Tdp1 double mutant strain has substantial topoisomerase-mediated DNA dam- (tyrosyl DNA phosphodiesterase I) by camptothecin sensitivity. The two genes age: nucleases and other phosphodi- Nash and colleagues [6, 7]. Subsequent identified in the screen were Tdp1 and esterases. Nucleases cut DNA internal work demonstrated that this protein was TTRAP. As expected from the genetic to a trapped topoisomerase, while found in all eukaryotes, and showed screen, purified TTRAP protein can phosphodiesterases specifically cleave that the enzyme was a phospholipase process Top1-mediated DNA damage, the phosphotyrosine linkage initially D superfamily enzyme [8, 9]. While although it processes 3′ phosphotyrosyl formed during topoisomerase-mediated the initial characterization of the yeast moieties rather inefficiently. TTRAP is cleavage of DNA. Several nucleases enzyme suggested that Tdp1 was substantially more active with 5′ phos- specific for 3′ damage to DNA, and photyrosyl modified oligonucleotides as could disjoin Top1 trapped on DNA, a substrate. Importantly, siRNA knock- Correspondence: John L Nitiss subsequent studies demonstrated that down of TTRAP results in sensitivity Tel: +901-595-2794; Fax: +901-595-4290 yeast Tdp1 also protected cells from to the Top2 targeting agent etoposide, E-mail: [email protected] Top2-mediated DNA damage, and could but not to the Top1 targeting agent

Cell Research | Vol 20 No 2 | February 2010 npg 123 camptothecin. Consistently, overexpres- provided support for the hypothesis 1996; 93:11534-11539. sion of TTRAP in yeast cells confers that Tdp1 represents one of many DNA 8 Davies DR, Interthal H, Champoux JJ, resistance to etoposide compared to repair functions that are neuroprotec- Hol WG. Insights into substrate binding and catalytic mechanism of human ty- wild type cells. These results strongly tive [15]. Since TTRAP was originally rosyl-DNA phosphodiesterase (Tdp1) implicate TTRAP as an important activ- identified as a protein associated with from vanadate and tungstate-inhibited ity for repairing Top2-mediated DNA TNF receptor family members, and structures. J Mol Biol 2002; 324:917-932. damage [12]. plays an important role in Zebrafish 9 Davies DR, Interthal H, Champoux JJ, Repair of either Top1- or Top2-medi- embryogenesis [16, 17], complete loss- Hol WG. The crystal structure of hu- ated damage is a complicated biological of-function mutations may be difficult man tyrosyl-DNA phosphodiesterase, process. The first step requires recog- to study in mouse systems. Nonetheless, Tdp1. Structure 2002; 10:237-248. nition that the enzyme is “trapped”. once they are better understood, the 10 Nitiss KC, Malik M, He X, White SW, Nitiss JL. Tyrosyl-DNA phosphodiesterase This recognition probably relies on the pathways that include TTRAP should (Tdp1) participates in the repair of Top2- interference of the trapped complex illuminate the repair of topoisomerase mediated DNA damage. Proc Natl Acad with ongoing DNA metabolic events mediated damage, and perhaps, lead to a Sci USA 2006; 103:8953-8958. such as replication or transcription. In better understanding of how DNA repair 11 Interthal H, Chen HJ, Champoux JJ. dividing cells, recognition of trapped functions are required during neuronal Human Tdp1 cleaves a broad spectrum complexes is associated with replica- development. of substrates, including phosphoamide tion fork collisions. For non-replicating linkages. J Biol Chem 2005; 280:36518- 36528. cells, collision with transcribing RNA References 12 Ledesma FC, El Khamisy SF, Zuma polymerase is likely to be important. MC, Osborn K, Caldecott KW. A hu- 1 Nitiss JL. DNA topoisomerase II and After recognition of a trapped complex, man 5′-tyrosyl DNA phosphodiesterase its growing repertoire of biologi- an initial repair event may include that repairs topoisomerase-mediated DNA cal functions. Nat Rev Cancer 2009; proteasome-mediated degradation of damage. Nature 2009; 461:674-678. 9:327-337. the topoisomerase [13]. The proteoly- 13 Zhang AL, Lyu YL, Lin CP et al. A pro- 2 Nitiss JL. Targeting DNA topoisomerase tease pathway for the repair of topoi- sis is critical for several reasons. First, II in cancer chemotherapy. Nat Rev Can- somerase II-DNA covalent complexes. once degradation of the trapped protein cer 2009; 9:338-350. J Biol Chem 2006; 281:35997-36003. commences, the enzyme probably will 3 Pommier Y, Barcelo JM, Rao VA et al. 14 Takashima H, Boerkoel CF, John J Repair of topoisomerase I-mediated DNA be unable to reseal the topoisomerase et al. Mutation of TDP1, encoding a damage. Prog Nucleic Acid Res Mol Biol induced break. Therefore, the initiation topoisomerase I-dependent DNA dam- 2006; 81:179-229. of proteolysis commits the cell to repair age repair enzyme, in spinocerebellar 4 Keeney S, Giroux CN, Kleckner N. Mei- the topoisomerase mediated damage. ataxia with axonal neuropathy. Nat osis-specific DNA double-strand breaks Genet 2002; 32:267-272. Second, proteolysis cannot completely are catalyzed by Spo11, a member of a 15 Katyal S, El-Khamisy SF, Russell HR remove the protein. A phosphotyrosyl widely conserved protein family. Cell et al. TDP1 facilitates chromosomal adduct will remain bound to DNA, 1997; 88:375-384. single-strand break repair in neurons 5 Hartsuiker E, Neale MJ, Carr AM. Dis- requiring enzymes such as Tdp1 and and is neuroprotective in vivo. EMBO tinct Requirements for the Rad32(Mre11) TTRAP to completely remove the pro- J 2007; 26:4720-4731. Nuclease and Ctp1(CtIP) in the Removal tein. Proteolysis is important since Tdp1 16 Esguerra CV, Nelles L, Vermeire L et of Covalently Bound Topoisomerase I is relatively inactive against intact Top1 al. Ttrap is an essential modulator of and II from DNA. Mol Cell 2009; 33:117- Smad3-dependent Nodal signaling dur- trapped on DNA. It will be interesting to 123. ing zebrafish gastrulation and left-right determine whether the TTRAP requires 6 Pouliot JJ, Yao KC, Robertson CA, axis determination. Development 2007; proteolysis of trapped topoisomerases. Nash HA. Yeast gene for a Tyr-DNA 134:4381-4393. A major reason why Tdp1 attracted phosphodiesterase that repairs topoi- 17 Pype S, Declercq W, Ibrahimi A et al. somerase I complexes. Science 1999; attention in addition to its unique enzy- TTRAP, a novel protein that associ- 286:552-555. matic activity was the recognition that ates with CD40, tumor necrosis factor 7 Yang SW, Burgin AB, Jr., Huizenga mutations in Tdp1 could lead to a human (TNF) receptor-75 and TNF receptor- BN et al. A eukaryotic enzyme that genetic disorder, SCAN1 (spinocer- associated factors (TRAFs), and that can disjoin dead-end covalent com- inhibits nuclear factor-κB activation. J ebellar ataxia with axonal neuropathy) plexes between DNA and type I topoi- Biol Chem 2000; 275:18586-18593. [14]. Transgenic mouse models have somerases. Proc Natl Acad Sci USA