DNA single-strand break repair and spinocerebellar ataxia with axonal neuropathy-1

Neuroscience 145 (2007) 1260 –1266

DNA SINGLE-STRAND BREAK REPAIR AND SPINOCEREBELLAR ATAXIA WITH AXONAL NEUROPATHY-1 S. F. EL-KHAMISYa,b AND K. W. CALDECOTTa*

Tyrosyl DNA phosphodiesterase 1 (TDP1) and spinocerebellar ataxia with axonal neuropathy (SCAN1)

a

Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK

b

Biochemistry Department, Faculty of Pharmacy, Ain Shams University, P.O.B. 11566, Cairo, Egypt

Disorders in DNA repair typically result in multiple pathological phenotypes, depending on the nature of the repair defect (de Boer and Hoeijmakers, 2000; O’Driscoll and Jeggo, 2006). For example, in addition to spinocerebellar ataxia, the DNA double-strand break (DSB) response-defective disorders ataxia telangiectasia (AT) and ataxia telangiectasia like disorder (ATLD) exhibit radiosensitivity, immunodeficiency, and chromosomal instability (Stewart et al., 1999; Taylor et al., 2004; O’Driscoll and Jeggo, 2006). In contrast, Takashima et al. (2002) recently identified an autosomal recessive ataxia that they denoted SCAN1, the phenotype of which lacks chromosomal instability and cancer predisposition and appears to be restricted to the nervous system. SCAN1 is associated with a mutation in TDP1, a protein that is primarily involved in the repair of DNA strand breaks created by topoisomerase 1 (Top1). Top1 relaxes superhelical tension in DNA and during its normal catalytic cycle generates a reversible and transient intermediate known as the Top1 cleavage complex, in which Top1 is covalently attached via a tyrosyl residue (human Tyr723) to the 3=-terminus of a single-stranded nick (Wang, 2002). Following release of torsional stress, Top1 reseals the nick and restores the integrity of the double helix (Pommier et al., 2003). The rate of religation is normally much faster than the rate of cleavage and thus the steady state concentration of these intermediates is very low (Pommier et al., 2003; Rideout et al., 2004). However, under certain circumstances, Top1 cleavage complexes may become irreversible, most likely due to misalignment of the 5=-hydroxyl that is no longer able to act as a nucleophile in the religation reaction (Pommier et al., 2003). Consequently, this irreversible “abortive” Top1-associated DNA break requires a DNA repair process for its removal. For example, Top1 cleavage complexes can be converted into abortive Top1-associated DNA DSBs by collision with a replication fork (Avemann et al., 1988; Holm et al., 1989; Hsiang et al., 1989; Ryan et al., 1991; Strumberg et al., 2000). In addition, collision with the transcription machinery converts Top1 cleavage complexes into abortive Top1-associated DNA SSBs, and occasionally abortive Top1-associated DSBs if two Top1 cleavage complexes are closely spaced on opposite strands of DNA (Kroeger and Rowe, 1989; Bendixen et al., 1990; Wu and Liu, 1997). Finally, some mutagenic DNA adducts (Pourquier et al., 1998, 2001; Pommier et al., 2000a) and en-

Abstract—DNA single-strand breaks (SSBs) are the commonest DNA lesions arising spontaneously in cells, and if not repaired may block transcription or may be converted into potentially lethal/clastogenic DNA double-strand breaks (DSBs). Recently, evidence has emerged that defects in the rapid repair of SSBs preferentially impact the nervous system. In particular, spinocerebellar ataxia with axonal neuropathy (SCAN1) is a human disease that is associated with mutation of TDP1 (tyrosyl DNA phosphodiesterase 1) protein and with a defect in repairing certain types of SSBs. Although SCAN1 is a rare neurodegenerative disorder, understanding the molecular basis of this disease will lead to better understanding of neurodegenerative processes. Here we review recent progress in our understanding of TDP1, single-strand break repair (SSBR), and neurodegenerative disease. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

DNA is under continuous threat and thousands of DNA single-strand breaks (SSBs) arise in each cell per day (Lindahl, 1993). SSBs can arise either directly (e.g. from attack of deoxyribose by reactive oxygen species) or indirectly (e.g. via enzymatic cleavage of the phosphodiester backbone during DNA base excision repair). Cells have employed different mechanisms to repair DNA SSBs, which are collectively termed DNA single-strand break repair (SSBR) (Thompson and West, 2000; Caldecott, 2004). Defects in the repair of, or response to, DNA damage have been associated with many human disorders such as cancer, immunodeficiency, and neurodegeneration. Recent data suggest that defects in the repair or response to DNA SSBs may have particular impact on the nervous system, and are associated with spinocerebellar ataxia (Date et al., 2001; Moreira et al., 2001; Takashima et al., 2002).

*Corresponding author. Tel: ⫹44-01273-877511; fax: ⫹44-01273678121. E-mail address: [email protected] (K. Caldecott). Abbreviations: CPT, camptothecin; DSB, double-strand break; DSBR, double-strand break repair; HR, homologous recombination; LCL, lymphoblastoid cell line; SCAN1, spinocerebellar ataxia with axonal neuropathy; SSB, single-strand break; SSBR, single-strand break repair; TDP1, tyrosyl DNA phosphodiesterase 1; Top1, topoisomerase 1.

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.08.048

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dogenous DNA lesions such as abasic sites, nicks, gaps, and DNA secondary structures can convert Top1 cleavage complexes into abortive Top1-associated DNA SSBs (Fig. 1) (Lanza et al., 1996; Pourquier et al., 1997a,b; Christiansen and Westergaard, 1999; Pommier et al., 2000b). In 1996, Nash and colleagues (Yang et al., 1996) employed a single-stranded DNA oligonucleotide linked to a tyrosine residue via a 3=-phosphoryl group, mimicking an abortive Top1-associated DNA strand break, to identify an activity in Saccharomyces cerevisiae extract that was able to remove Top1 from the 3=-terminus of DNA. They named the activity Tdp1, and the gene encoding this activity was subsequently cloned (Pouliot et al., 1999). Tdp1 is highly conserved in eukaryotes, with identifiable orthologs in a wide variety of animals, including humans (Pouliot et al., 1999; Cheng et al., 2002; Pommier et al., 2003; Connelly and Leach, 2004; Davies et al., 2004). Sequence analysis of S. cerevisiae Tdp1 (Pouliot et al., 1999; Interthal et al., 2001) and the crystal structure of human TDP1 (Davies et al., 2002, 2003) reveal it

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to be a member of the phospholipase D (PLD) superfamily. These enzymes contain two copies of a highly conserved sequence (HXK(X)4D(X)6GSXN), known as HKD motif (Interthal et al., 2001) and catalyze phosphoryl transfer reactions. Despite lacking the aspartate conserved in other HKD motifs, the crystal structure of human TDP1 has shown that both histidine and lysine residues of the HKD motifs are clustered in the active centre of the enzyme (Davies et al., 2002, 2003, 2004). Our initial understanding of the role for TDP1 in DNA repair came from studies conducted in budding yeast. Yeast Tdp1 provides one of several potentially redundant mechanisms for removing Top1 peptide from a DSB created by collision of Top1 cleavage complexes with DNA replication forks (Pouliot et al., 2001; Vance and Wilson, 2002; Deng et al., 2005). Once Top1 peptide has been removed, the DSB is then channeled into the homologous recombination (HR) pathway for double-strand break repair (DSBR) (Pouliot et al., 2001; Vance and Wilson,

Fig. 1. Model for the generation of DNA-strand breaks from Top1 cleavage complexes. (A) Top1 relaxes torsional stress in DNA by introducing a reversible and transient DNA nick in which Top1 is covalently attached to the 3=-end. CPT reversibly inhibits the ligation step of Top1, thereby increasing the half-life of Top1 cleavage complexes. Note that some endogenous DNA lesions can also stabilize Top1 cleavage complexes (e.g. base mismatches) or induce their formation (e.g. 8-oxoguanine). (B) Top1 cleavage complexes can be converted into irreversible DNA DSBs by collision with a replication fork, or DNA SSBs by collision with the transcriptional machinery or by proximity to some types of DNA lesions.

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2002). Tdp1 in yeast appears to operate in conjunction with HR to repair Top1-associated DSBs, because mutation of Tdp1 is epistatic to mutations in critical components of the HR pathway, such as Rad52 (Pouliot et al., 2001; Vance and Wilson, 2002). As in yeast, it is likely that human TDP1 plays a role during DNA replication. The cytotoxic effect of the Top1 specific inhibitor camptothecin (CPT) is S-phase specific in most wild-type mammalian cell lines examined (Horwitz and Horwitz, 1973; D’Arpa et al., 1990; Ryan et al., 1994; Poot et al., 1999), and is ablated by co-incubation with aphidicolin, an inhibitor of DNA replication (Holm et al., 1989; Ryan et al., 1991). Thus, ongoing replication forks appear to be required to convert Top1 cleavage complexes into cytotoxic DSBs, in wild type proliferating cells at least (Shao et al., 1999; Arnaudeau et al., 2001; Culmsee et al., 2001; Godthelp et al., 2002; Wu et al., 2002; Furuta et al., 2003; Hinz et al., 2003; van Waardenburg et al., 2004). It is therefore plausible that TDP1 inhibitors may enhance the anti-cancer activity of Top1 inhibitors and act as anti-proliferative agents (Liao et al., 2006). Consistent with a possible role for human TDP1 during replication, lymphoblastoid cell lines (LCLs) from affected SCAN1 individuals appear to show more late S-phase arrest than normal cells following CPT treatment (Interthal et al., 2005b). This could reflect a direct role for TDP1 during replication or an accumulation of un-repaired SSBs that are consequently converted into DSBs by collision with replication forks. Despite its pivotal role in the repair of certain types of replication-dependent DSBs it seems unlikely that this can account for the pathology of SCAN1, which appears to be restricted to post-mitotic neurons and lacks any obvious sign of genetic instability. It is possible that SCAN1 results from a defect in the repair of replication-independent DSBs, which might arise from overlapping Top1-SSBs for example. However, since (like other SSBs) the occurrence of Top1-SSBs is most likely several orders of magnitude more frequent than Top1-DSBs, and since SSBs can block transcription (Kroeger and Rowe, 1989; Bendixen et al., 1990; Zhou and Doetsch, 1994; Wu and Liu, 1997), it seems plausible that a defect in the repair of SSBs might account for the neurodegenerative pathology in SCAN1. Consistent with this idea, whereas we have failed to observe a measurable defect in the repair of replicationindependent DSBs in CPT-treated SCAN1 LCLs, we have observed a profound defect in their ability to repair replication-independent SSBs. Intriguingly, SCAN1 cells were also less able than normal cells to repair oxidative DNA SSBs induced by H2O2, though the defect was not as pronounced as for CPT (El-Khamisy et al., 2005). This could reflect a direct requirement for TDP1 in processing oxidative DNA breaks, since it has been shown that TDP1 can remove glycolate from 3=-phosphoglycolate termini of strand breaks (Inamdar et al., 2002) and a variety of other 3=-adducts from DNA (Interthal et al., 2005a). In addition, yeast expressing an active site mutant of Tdp1 (His182Ala) is hypersensitive to bleomycin, a radiomimetic agent which generates DNA breaks that are almost exclusively terminated with 3=-phosphoglycolate (Liu et al., 2004). More-

over, in vitro assays have recently shown that SCAN1 cell extracts lack any detectable processing of 3=-phosphoglycolate at single-stranded oligomers or at 3=-overhangs of DSBs (Zhou et al., 2005). However, similar assays with substrates that mimic oxidative SSBs suggest that APE1 is the major 3=-phosphoglycolate-processing activity in human cell extracts (Parsons et al., 2004). This suggests that TDP1 may be a major contributor in removing 3=-phosphoglycolate at sites of DSBs, but not at SSBs. An alternative explanation for the role of TDP1-dependent SSBR at oxidative SSBs would be an increased formation of Top1-associated SSBs following treatment with H2O2. Indeed, the presence of nicks (Pourquier et al., 1997a), abasic sites (Pourquier et al., 1997b), modified bases (Pourquier et al., 1999; Lesher et al., 2002), and modified sugars (Chrencik et al., 2003) has been shown to stabilize and/or induce Top1 cleavage complexes. Consistent with this, Top1-deficient P388/CPT45 murine leukemia cells are more resistant to H2O2 than Top1-proficient parental cells (Daroui et al., 2004). In addition, over-expression of Top1 in yeast confers sensitivity to H2O2 and top1-mutant strains are more resistant to H2O2 than their isogenic wild-type strains (Daroui et al., 2004). Finally, studies employing HeLa cells have confirmed that H2O2 does indeed induce Top1-DNA lesions (Daroui et al., 2004). Yeast two-hybrid and coimmunoprecipitation experiments have provided evidence for a direct interaction between TDP1 and DNA ligase III␣, a component of the DNA SSBR machinery (El-Khamisy et al., 2005). In agreement with this, it has been shown that TDP1 co-immunoprecipitates with XRCC1 from rodent cell extract (Plo et al., 2003). The interaction between TDP1 and DNA ligase III␣ is mediated by the N-terminal domain of TDP1, a region that is poorly conserved or absent from Tdp1 of lower eukaryotes (Interthal et al., 2001; El-Khamisy et al., 2005). Budding yeasts lack orthologs of various components of SSBR machinery found in mammals, including XRCC1 and DNA ligase III␣. Therefore it seems likely that the N-terminal domain of human TDP1 may have evolved to couple this enzyme to the SSBR machinery, through an interaction with DNA ligase III␣. Current models of SSBR suggest sequential recruitment of the enzymes such that DNA repair-intermediates are passed from one enzyme to the other in a molecular relay (Mol et al., 2000; Wilson and Kunkel, 2000). However, at least some of the XRCC1 appears to be present in pre-formed multi-protein complexes (Vidal et al., 2001; Whitehouse et al., 2001; Caldecott, 2003; Clements et al., 2004), though it is unclear whether these interactions are occurring in response to “endogenous” levels of DNA damage or are truly constitutive. It should be noted that although we have not yet detected a measurable defect in the repair of replicationindependent DSBs in SCAN1 cells following CPT or oxidative stress, it is possible and indeed perhaps likely that one exists. Nevertheless, the observations that SSBs can be potent blocks to transcription and that they arise several orders of magnitude more frequently than DSBs renders

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these lesions a likely threat to cellular function, if they are not repaired rapidly. SCAN1 and neurodegeneration It is currently unclear why loss of TDP1 does not result in increased genetic instability and cancer. However, a likely contributing factor is the ability of alternative end processing proteins such as ERCC1/XPF, in conjunction with HR repair, to compensate for defective TDP1-SSBR by removing un-repaired SSBs during DNA replication. Consistent with this, HR is selectively elevated in SCAN1 cells as indicated by an increased frequency of “spontaneous” and “CPT-induced” sister chromatid exchanges (El-Khamisy et al., 2005). Thus, it seems possible that the concerted activity of structure specific nucleases and the HR machinery can fulfill a backup role in the absence of TDP1-

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dependent SSBR, thereby allowing proliferating SCAN1 cells to tolerate un-repaired SSBs, at physiological levels of DNA damage at least. This scheme is also applicable to other types of un-repaired SSBs, since structure specific nucleases may enable removal of a wide variety of damaged 5= and 3=-termini (Fig. 2). It is also currently unclear why the nervous system might be so sensitive to loss of TDP1, compared with other largely non-cycling tissues for example. One likely factor is that the nervous system consumes ⬃20% of inhaled oxygen and possesses low levels of antioxidant enzymes (Barzilai et al., 2002), and so encounters high levels of oxidative stress. Consequently, since TDP1 may be required for the repair of SSBs and DSBs arising either directly or indirectly through oxidative damage, the nervous system may be particularly dependent on TDP1. In

Fig. 2. Model for the impact of SSBs on proliferating and non-proliferating cells in the absence of SSBR. In both non-cycling and cycling cells SSBs are normally rapidly repaired by SSBR. However, in SSBR-defective non-cycling cells (left), SSBs persist and block transcription leading to cell death. Compared with other largely non-cycling tissues, we propose that the elevated oxidative stress, increased transcriptional demand, and limited cellular regenerative capacity in the nervous system render this tissue particularly dependent upon SSBR. In cycling cells lacking SSBR (right) un-repaired SSBs are converted into DSBs by collision with DNA replication forks (red arrow). Structure-specific 5= and 3=-nucleases can then process the damaged 5=-and 3=-termini (yellow ovals) enabling the DSB to be faithfully repaired by HR. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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addition, there is a high transcriptional demand in postmitotic neurons. For example, cortical neurons incorporate ⬃5.5-fold more 3H-uridine than astrocytes (Morris and Geller, 1996) and possess twice the number of transcription initiation sites as compared with glial cells (Flangas and Bowman, 1970; Sarkander and Uthoff, 1976; Sarkander and Dulce, 1978), and un-repaired SSBs can be potent blocks to RNA polymerases, which may subsequently trigger cell death. The limited regenerative capacity of the nervous system, compared with other predominantly non-cycling tissues, may also be a factor, and may render this tissue particularly susceptible to cell loss. In contrast, cell loss from tissues with greater regenerative capacity may be better tolerated. For example, myocytes and adipocytes are also terminally differentiated, but can be replaced by precursor cells (Vierck et al., 2000; Yan, 2000; Nouspikel and Hanawalt, 2002).

CONCLUSIONS Whereas great insights into the relationship between TDP1, DNA strand break repair, and neurodegeneration have emerged, there is still much that is unresolved. For example, what are the physiologically relevant substrates fro Tdp1 in vivo. In addition to 3=-termini created by Top1 and oxidative damage, recent data suggest that Tdp1 may also operate on certain types of 5=-termini (Nitiss et al., 2006), increasing the types of lesion that may impact on neurological integrity in SCAN1 individuals. Also, why does loss of Tdp1 selectively affect certain neuronal tissue such as the cerebellum, and what is the relative contribution of SSBs versus DSBs in neurodegeneration? The development of animal models with specific defects in SSBR and DSBR will be an important tool to address these questions. Acknowledgments—We apologize to colleagues whose primary research papers may not have been cited due to space constraints. This work was supported by an MRC programme grant to K.W.C. and an ORS to S.F.E-K.

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(Accepted 7 August 2006) (Available online 11 October 2006)