Repair of DNA Covalently Linked to Protein

Molecular Cell, Vol. 13, 307–316, February 13, 2004, Copyright 2004 by Cell Press

Repair of DNA Covalently Linked to Protein John C. Connelly* and David R.F. Leach Institute of Cell and Molecular Biology University of Edinburgh Kings Buildings Edinburgh EH9 3JR United Kingdom

Summary A potentially lethal form of DNA/RNA modification, a cleavage complex, occurs when a nucleic acid-processing enzyme that acts via a transient covalent intermediate becomes trapped at its site of action. A number of overlapping pathways act to repair these lesions and many of the enzymes involved are those that catalyze recombinational-repair processes. A protein, Tdp1, has been identified that reverses cleavage-complex formation by specifically hydrolyzing a tyrosyl-DNA phosphodiester bond. The study of these pathways is both interesting and pertinent as they modulate the effectiveness of many antitumor/antibacterial drugs that act by stabilizing cleavage-complexes in vivo. Introduction A number of nucleic acid processing enzymes form a highly reactive intermediate between their substrate and a specific amino acid residue located in the enzymes’ active site. These enzymes act via a two-step reaction cycle in which two sequential attacks generate a covalent intermediate that is then cleaved to release a final product. This is an extremely hazardous mode of catalysis for an enzyme involved in nucleic acid metabolism as any failure in the release step of the cycle can result in a remarkable form of self-inflicted nucleic acid damage and the formation of a long-lived intermediate known as a covalent complex, or cleavage complex. These lesions can be converted to more permanent single-strand breaks (SSBs), or double-strand breaks (DSBs) via the action of RNA transcription and DNA replication machinery (Hong and Kreuzer, 2003, and within). In this review we focus primarily on the pathways that act to repair topoisomerase-mediated DNA damage in vivo as a number of recent publications are beginning to illuminate the systems employed to deal with these particular lesions. It is likely however that the pathways and strategies employed to repair topoisomerase damage can be deployed on covalent complexes generated by other, similarly acting, enzymes. Cellular Enzymes with the Potential to Form Covalent Complexes In Vivo The serine/tyrosine recombinase family of proteins has come to typify nucleic acid processing enzymes that act via a transient covalent intermediate. These include the site-specific recombinases of prokaryotes and yeast *Correspondence: [email protected]

Review

(e.g., bacteriophage ␭ Integrase, XerCD of Escherichia coli, and Flp of Saccharomyces cerevisiae) and the topoisomerases present in all living organisms. Topoisomerases are essential cellular enzymes required to control the superhelical tension and DNA entanglement associated with DNA replication, recombination, transcription, and chromosome segregation (Wang, 1996). In eukaryotes the enzyme DNA topoisomerase I breaks one strand of a DNA duplex, then relieves torsional stress before catalyzing the rejoining of the broken strand. This reversible breaking and joining reaction is achieved via an intermediate that consists of a tyrosine residue of topoisomerase I covalently linked to broken DNA, via a 3⬘-phosphoryl group. This covalent protein-DNA complex (cleavage complex) is usually transient and is resolved in the resealing step of the reaction. However, if the rejoining step fails, the result is a protein-bound discontinuity in the backbone of DNA (Figure 1Ai). It is known that imperfections in the structure of DNA, such as base mismatches, gapped and nicked molecules, and a variety of DNA adducts (all of which can arise via the action of endogenously and exogenously acting agents such as reactive oxygen and/or ionizing radiation) promote the formation of long-lived topoisomerase-cleavage complexes (Yang et al., 1996; Debe´thune et al., 2002). Like topoisomerase I, members of the topoisomerase II family of enzymes can also form cleavage complexes via a tyrosyl-or seryl-DNA phosphodiester bond. However, the covalent linkage in this instance is formed at the 5⬘-end of a DNA molecule, rather than the 3⬘-end, and occurs on both strands of a duplex (Figure 1Aii). Thus, covalent complexes generated by the type I and II topoisomerases are chemically very different. As topoisomerases are required particularly in actively growing cells, they have become targets for many cytotoxic drugs. Several potent antibacterial and anticancer drugs act by inhibiting the resealing step of topoisomerases and their efficacy is achieved by promoting the formation of these toxic lesions (Chen and Liu, 1994). The commonly used antibiotic nalidixic acid and the chemotherapeutic drug camptothecin (CPT) act in this way. While much is known of the pathways that act to repair SSBs and DSBs, little is known about the systems that act to repair DNA molecules containing covalently bound protein molecules. Understanding the relationships and mechanics of these largely uncharacterized systems is extremely important as it will aid the design of more effective antibacterial and chemotherapeutic regimes. In addition to the serine/tyrosine recombinase family, several other DNA processing enzymes act via covalent intermediates, and their covalent complexes have been trapped in vitro. For example, the ubiquitous DNA repair enzymes Endonuclease III and related enzymes (such as human 8-oxoguanine glycosylase, hOgg1; Endonuclease VIII; and MutY) are DNA glycosylases that remove oxidized-pyrimidine lesions using an active site lysine (Eisen and Hanawalt, 1999). These proteins form Schiff base-intermediates that have been trapped in vitro

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Figure 1. Groups Found at DNA Termini (A) Intermediates of topoisomerase reactions. (i) Topoisomerase I-DNA cleavage complex. Nicked duplex molecule (blue) with topoisomerase I covalently linked to the 3⬘phosphoryl group of one strand (at the site of the break) via a tyrosine molecule. P, phosphoryl group; Y, tyrosine. Topoisomerase I is shown as a red circle. (ii) Topoisomerase IIDNA cleavage complex. A DNA duplex (blue) covalently linked, via a 5⬘-phosphoryl group, to (in this instance) a tyrosine group of topoisomerase II. P, phosphoryl group; Y, tyrosine. Topoisomerase II is shown as two red circles. (B) Reaction performed by DNA ligase. Two ssDNA molecules that lie adjacent to each other in a DNA duplex are shown (only one of the complementary strands of a duplex is drawn). One of the adjacent strands terminates with a 3⬘-hydroxyl residue (blue) and the other a 5⬘-phosphate group (red). DNA is shown as an arrowheaded line with 5⬘- to 3⬘polarity. The product of the DNA ligase reaction is seen as a hybrid blue/red molecule.

(Hilbert et al., 1996; Nash et al., 1997; Williams and David, 1998; Zharkov et al., 2002; Fromme and Verdine, 2003). The protein Tdp1 (that repairs type I topoisomerasecleavage complexes, see below) acts via a transient covalent intermediate and an hTdp1:DNA covalent complex has also been detected in vitro (Interthal et al., 2001). In many cases a failure in the release step of the reaction cycle of these enzymes can result in the formation of a more lethal DNA lesion than that which was being acted upon originally. Members of the DNA m5C methyltransferase family of DNA modifying enzymes alter their respective substrates by transferring a methyl group to a pyrimidine residue of a nucleotide (see Liu and Santi, 2000). They do this by forming a transient covalent intermediate between the pyrimidine ring of the substrate and the sulfur atom of a cysteine residue located in the enzyme’s active site. This intermediate is usually released by a second attack from a neighboring cysteine group. Covalent intermediates that use this reaction pathway have been trapped in vitro using chemically modified nucleotide substrates (King and Redman, 2002). Interestingly, RNA molecules also have the potential of being damaged by covalently bound protein as a number of RNA m5C methyltransferases and their cleavage complexes have

been identified (Liu et al.; 2002; King and Redman, 2002). The Nop2 protein of S. cerevisiae is involved in ribosomal RNA processing and contains the two active site cysteine residues associated with the RNA/DNA-m5C methyltransferase family. It has been postulated that Nop2 acts via a transient covalent intermediate, formed by a primary cysteine and then released by a secondary cysteine. A slowly migrating Nop2 species (presumably a Nop2:RNA cleavage complex) is observed when the cysteine residue involved in the release step of the reaction cycle is mutated. An RNA:protein cleavage complex has been shown to be formed when the similarly acting yeast tRNA-methyl transferase, Ncl1, is mutated in the analogous cysteine residue (King and Redman, 2002). No such complexes are seen when the cysteine involved in the primary step of catalysis was mutated. It is interesting to note that yeast mutants containing a mutation in the cysteine associated with intermediate release are nonviable, whereas mutations in the cysteine associated with the primary step (and double mutants) are viable (King and Redman, 2002). Therefore, failure to release a covalently bound protein from a RNA molecule in vivo can prove lethal. All of the aforementioned enzymes have the potential to form toxic lesions. However, their frequency of cova-

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lent complex formation in vivo remains to be investigated. Clearly, for the topoisomerases, cleavage complex formation increases due to the action of intracellular and extracellular DNA damaging agents and various drugs (see above). Also, mutant forms of topoisomerase that are inefficient in the release step of the catalytic cycle result in increased complex formation in vivo in the absence of drugs (Pouliot et al., 1999). It has been demonstrated that mutations in trans-acting genes can also increase the frequency of topoisomerase-covalent complex in vivo. For example, in S. cerevisiae the life span of the Spo11-cleavage complex (Spo11 generates the DSBs employed to initiate meiotic recombination) is enhanced by mutation of the MRE11 and/or RAD50 genes (see below) The Tdp1 Protein: An Enzyme that Disjoins Tyrosine-DNA Covalent Complexes All DNA repair pathways require that the 3⬘- and 5⬘termini of a broken strand be suitable substrates for DNA ligase (Figure 1B). A protein-DNA cleavage complex therefore presents an obstacle to the cell’s DNA repair machinery. It renders a DNA end inaccessible to repair ligases and polymerases, and therefore a covalent complex must be processed to ensure successful repair. Yang et al. (1996) used a single-strand (ss)-DNA oligonucleotide linked to a tyrosine residue via a 3⬘-phosphoryl group (mimic of a topoisomerase cleavage complex— see Figure 2A) to identify an activity in S. cerevisiae cell extracts that was able to remove a topoisomerase I-covalent complex from a DNA end. This activity could remove, specifically, the tyrosine group from the covalent complex by hydrolyzing the phosphodiester bond between the tyrosine group and the terminal 3⬘-phosphate of the oligonucleotide (Figure 2A). They named the activity Tdp1 (Tyrosyl-DNA Phosphodiesterase 1). In addition to ssDNA substrates, Tdp1 is active on tyrosine-oligonucleotides containing duplex DNA (Figure 2B and below). It is also capable of removing an entire protein from the 3⬘-end of a duplex, i.e., when a similar substrate containing bacteriophage ␭ integrase was incubated with Tdp1, the protein was removed from the 3⬘-end of the substrate. Tdp1 action is absolutely specific for 3⬘-phosphodiester linkages, and does not cleave 5⬘-phosphodiester linkages (Yang et al., 1996). Tdp1 Sequence Analysis Nash and colleagues identified the gene encoding Tdp1 by assaying extracts of mutagenized yeast cells (Pouliot et al., 1999). Sequence analysis of S. cerevisiae Tdp1 (scTdp1) reveals it to be a member of the Phospholipase D (PLD) superfamily of proteins. These enzymes contain two copies of the highly conserved sequence HXK(X)4D(X)6GSXN, known as an HKD motif (Interthal et al., 2001, and within), and most share a common reaction chemistry, i.e., they catalyze a phosphoryl transfer reaction. The crystal structures of various members reveal that both histidine and lysine residues of the HKD motifs are clustered in the active center of the enzymes. Within the PLD superfamily, Tdp1 forms a separate class, as it possesses a unique HKD motif in that it lacks an aspartate residue conserved in other PLD family members. Human Tdp1 (hTdp1) also is a PLD superfamily

member that acts via the same mechanism as scTdp1 (Interthal et al., 2001). These observations suggest that the HKD motifs of Tdp1 proteins are involved in catalysis and act in a similar manner to the other PLD superfamily members.

Tdp1 Substrate Preference The nature of the substrate(s) that Tdp1 acts upon in vivo is not known. However, biochemical and genetic studies are revealing the type of structures Tdp1 might recognize within a cell. Native scTdp1 possesses no 3⬘- or 5⬘-phosphatase activity and has a much higher specificity for a tyrosine-phosphodiester bond than any other phosphodiester bonds present in synthetic cleavage complexes (Yang et al. 1996). These observations indicate that scTdp1 recognizes the chemistry of a tyrosyl-phosphodiester linkage rather than any structural feature of a phosphodiester bond. When recombinant scTdp1 was incubated with a number of synthetic substrates (designed to mimic structures that the enzyme might encounter in vivo—see Figure 2B), it always cleaved a tyrosyl-phosphodiester bond to yield an oligonucleotide product ending with a 3⬘-phosphoryl group. Single-strand, blunt-end duplex and tailed duplex substrates (containing a tyrosine molecule at a 3⬘-terminal position) were cleaved to the same extent. However, nicked-duplex substrate was cleaved to a lesser degree. These observations suggest that scTdp1 acts preferentially on topoisomerase I-cleavage complexes after they have been converted to doublestrand breaks (DSBs) by the replication machinery of a cell. This proposal is substantiated by the finding that Tdp1-mediated repair of a topoisomerase I cleavage complex occurs via a DSBR pathway in S. cerevisiae, i.e., is RAD52 dependent (Pouliot et al., 2001; and see below). It is known that DNA replication enhances the cytotoxic effects of topoisomerase I inhibitors such as CPT (Nitiss and Wang, 1988; Eng et al., 1989). All of these observations suggest that the nicked molecules generated by topoisomerase I are converted to DSB structures (presumably by the DNA replication apparatus) with a topoisomerase molecule trapped at the end of the duplex. The substrate Tdp1 acts upon is unusual in that it contains both a protein and nucleic acid component, which happen to be covalently linked to each other. ScTdp1 prefers nucleopeptides with shorter amino acid lengths and longer oligonucleotide tracts. In addition, a topoisomerase I-cleavage complex composed of a 70 kDa fragment of human topoisomerase I, linked to a duplex suicide substrate, is a substrate for Tdp1; however, it is not a particularly good one. These observations suggest that the topoisomerase component of the complex may undergo some form of unfolding and/or proteolysis prior to Tdp1 action in vivo (Debe´thune et al., 2002), and that binding to the DNA component of a cleavage complex is important for scTdp1 activity. Taken together, all of these observations suggest that, in S. cerevisiae, Tdp1 acts on a protein-DNA structure that has been generated after DNA replication, where the peptide component has undergone some form of reorganization.

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Figure 2. Substrates Used to Identify and Characterize Yeast Tyrosyl-DNA Phosphodiesterase (Tdp1) Activity (A) Tyrosyl-oligonucleotide substrate used to identify scTdp1 activity and the product generated by it. The oligonucleotide component of the substrate is shown as a blue arrowheaded line with 5⬘- to 3⬘-polarity. The tyrosine component of the substrate is linked to nucleic acid via a 3⬘-phosphodiester linkage and is shown as a red hexagon. 32P, position of radioactive label within molecule. The specific oligonucleotide product formed after incubation with Tdp1 with the tyrosyl group removed is shown below the tyrosyl-DNA substrate. (B) Other DNA molecules thought to mimic in vivo substrates that have been used to characterize Tdp1 activity. Column 1, molecules that are good to moderate substrates for Tdp1; column 2, molecules that are weak/ slowly hydrolysable substrates for Tdp1; ssDNA, single arrowheaded molecule; DsDNA, double arrowheaded molecule; *, position of 32P-label; Y, tyrosine group; PG, phosphoglycolate moiety; Red circle, native protein molecule e.g., topoisomerase or ␭ integrase; green beads, synthetic peptide fragment. (i) ss-oligonucleotide with 3⬘-tyrosine; (ii) duplex; (iii) tailed duplex; (iv) nicked or gapped duplex; (v) tailed duplex with proteolyzed or denatured protein fragment attached at 3⬘-end; (vi) nucleopeptide substrate; (vii) duplex substrate with 3⬘-PG; (viii) substrate containing native protein; (ix) 5⬘linked tyrosine substrate, topoisomerase IIcleavage complex mimic; (x) blunt end duplex with no tyrosine; (xi) Rad1/10 flap substrate; (xii) Mus81/Mms4 branch substrate.

Tdp1 Structure and Mechanism of Action Recently, various hTdp1 crystal structures have been solved and are providing fascinating insights into the mechanics of Tdp1 action (Davies et al., 2002a, 2002b, 2003). The crystal structure of a quaternary-covalent complex of hTdp1 (that forms a mimic for the transition state of hTdp1 acting on a cleavage complex) has been solved (Davies et al., 2003). It consists of hTdp1, vanadate (a phosphate transition-state analog essential for obtaining the quaternary structure), an eight amino acid peptide (corresponding to a region of human topoisomerase I-that contains the active site tyrosine), and a six

base oligonucleotide (of a known topoisomerase I substrate). This structure reveals, very clearly, the different substrate binding regions within hTdp1 (Figure 3A). It demonstrates that the DNA moiety of the quaternary complex is bound in a narrow, positively charged substrate binding groove and that peptide binding occurs in a wider substrate binding cleft (Figure 3A). The conformation of the topoisomerase peptide seen in the quaternary complex is quite different to the corresponding region found within the crystal structures of human topoisomerase I-cleavage complexes. The tyrosyl-phosphodiester bond to be cleaved by Tdp1 is inaccessible and

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Figure 3. Tdp1 Structures (A) Electrostatic potential surface of hTdp1 showing sites of nucleotide and peptide interaction. The molecular surface is colored between -10kT (red) and ⫹10kT (blue). The peptide-vanadate-DNA substrate mimic is displayed as a stick structure. The yellow V indicates the position of the vanadate residue in the active site. The DNA moiety extends above the active site bound in the narrow, positively charged half of the substrate binding groove. The peptide moiety is located below the active site in a relatively neutral portion of the lower and wider substrate binding cleft. Image and legend reproduced from Davies et al. (2003). (B) Diagram showing the hydrogen bonding and electrostatic interaction patterns observed in the active site of wild-type hTdp1. (C) Hydrogen bonding and electrostatic interaction pattern observed in the active site of the His493Arg hTdp1 mutant. The side chain of the mutated arginine residue probably turns away from the active center and form electrostatic interactions with the adjacent carbonyl group of Ala287 and the carboxyl side chain of Asp 288. Compare B to C. Image and legend reproduced from Takashima et al. (2002) (http://www.nature.com).

buried deep within the topoisomerase I structure (Redinbo et al., 1998). It has been suggested that these observations provide evidence that the topoisomeraseDNA complex undergoes extensive structural changes (e.g., proteolysis and/or unfolding) prior to cleavage by Tdp1 (Davies et al., 2003). The overall conformation of the nucleic acid component of the quaternary complex is also distorted. It is irregular, extended and elongated compared to normal B form DNA. Therefore, the DNA binding region of hTdp1 is unlikely to accommodate duplex DNA. As phosphotyrosine at the 3⬘-end of a dsDNA (or ssDNA) molecule is a substrate for scTdp1, it would appear that a conformational change in scTdp1 must occur prior to catalysis, or the DNA undergoes some sort of melting event. Mutational analyses have revealed that the invariant histidines and lysines of the HKD motifs of hTdp1 are required for enzymatic activity as both of the active site histidines (His263 and His493) are absolutely essential

for catalysis (Interthal et al., 2001). The crystal structure of hTdp1 in complex with the phosphate transition state analogs vanadate and tungstate (that inhibit a variety of enzymes involved in phosphoryl transfer reactions by acting as transition state analogs) reveals that the inhibitors are covalently bound to His263 (Davies et al., 2002b, 2003). This confirms His263 as the more reactive of the two active site histidines and suggests that it acts as a nucleophile in the first step of the catalytic cycle to form the phosphoenzyme intermediate. The amino acid residues and steps involved in the second state of the reaction cycle are less clear. Tdp1 and the Genetic Disorder SCAN1 In eukaryotes, defects in a number of DNA repair pathways (e.g., DSBR, SSBR, nucleotide excision repair, base excision repair) result in genetic disease. Therefore it was not surprising to find that, in humans, mutation in Tdp1 is associated with a genetic disorder, spinocere-

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bellar ataxia with axonal neuropathy (SCAN1) (Takashima et al., 2002). Individuals with SCAN1 are characterized by possessing early onset cerebellar ataxia and axonal neuropathy. However, unusually for a defect in a DNA repair gene, they lack phenotypes such as genetic instability or cancer. SCAN1 is an autosomal recessive disorder that arises as a result of a histidine to arginine mutation in the conserved His493 residue of the second HKD motif of hTdp1 (Takashima et al., 2002). Protein modeling studies suggests the His493-Arg mutation most likely affects hTdp1 function by disrupting the symmetrical nature of the two conserved HKD motifs in the active site (compare Figures 3B to 3C). An attractive model of Tdp1 action (based largely on genetic observations made in yeast) suggests that the enzyme acts on dsDNA-cleavage complexes formed, via DNA replication, in rapidly replicating tissues. However, the most surprising observation about SCAN1 is that the cellular defect is most pronounced in terminally differentiated, nondividing tissues. These cells are characterized by having low levels of DNA replication and high levels of transcription. Repair Pathways for Topoisomerase I-DNA Covalent Complexes SSBR in Mammalian Cells Topoisomerase I action is often associated with RNA transcription (Wang, 1996) and therefore it has been suggested that, in mammals, Tdp1 acts via a SSBR pathway to repair SSBs associated with stalled transcriptional complexes (Takashima et al., 2002; Caldecott, 2003a and 2003b; Plo et al., 2003). Evidence for this hypothesis is provided by the following observations: mammalian cells containing the inactive SSBR proteins, PARP (polyADP-ribose polymerase) and XRCC1 (a scaffolding protein), both of which are components of a SSBR-repair complex, are hypersensitive to CPT. Also, cells overexpressing XRCC1 are more resistant to topoisomerase poisons (Park et al., 2002). In addition, the SCAN1 phenotype is most pronounced in terminally differentiated nondividing tissues that possess low levels of DNA replication but high levels of transcription. These observations suggest that SSBR pathways are the preferred route for repairing topoisomerase I-cleavage complexes in mammalian cells. Recently, Plo et al. (2003) demonstrated that Tdp1 interacts physically and functionally with members of the XRCC1-associated repair complex. They have proposed that Tdp1 hydrolyzes a tyrosyl-phosphodiester bond (formed between DNA and a topoisomerase molecule) generating a nucleotide product that terminates in a 3⬘-phosphoryl group. Polynucleotide kinase phosphatase (PNKP) then acts on this molecule to remove the 3⬘-phosphate moiety and phosphorylate the neighboring 5⬘-hydroxyl. Repair polymerases and ligases are then free to act upon these termini. Employing SSBR-proteins to repair cleavage complex damage does not exclude the possibility that DSBR pathways could also be used, perhaps in certain cells during the G2 and S phases of the cell cycle when sister chromatids are available for recombinational repair. However, SSBR has the advantage that DSBs are not generated, and thus it avoids the possibility of repair by nonhomologous end-joining pathways and the concomitant threat of genome rearrangement.

DSBR in Yeast Cells Yeast lacks orthologs of various components of the SSBR pathway found in mammals; consequently it may prefer to use Tdp1 via a DSBR pathway. Genetic analyses in S. cerevisiae indicate that at least two DSBR pathways exist to repair topoisomerase I damage (Pouliot et al., 2001). When the sensitivity of yeast cells to a mutant form of topoisomerase I (that results in increased cleavage complex formation) was examined, it was found that tdp1, rad52, and rad9 single mutants were sensitive to this particular form of lesion. However, a rad52 tdp1 double mutant is no more sensitive to toxic topoisomerase than a rad52 mutant alone. This observation indicates that RAD52 and TDP1 act in the same epistasis group and, in this context, Tdp1 in yeast functions as part of a DSBR pathway (Figure 4). A rad52 rad9 double mutant is no more sensitive to toxic topoisomerase than a rad52 mutant alone, indicating that these proteins operate in the same pathway. However, TDP1 and RAD9 are not in the same epistasis group, as tdp1 rad9 double mutants are more sensitive to CPT than either single mutant alone (Pouliot et al., 2001). These data indicate that Tdp1 and Rad9 both act via a DSBR pathway to correct topoisomerase damage in S. cerevisiae (Figure 4). In yeast, mutants defective in members of the RAD52 epistasis group (e.g., RAD50, RAD51, RAD52, RAD54, and MRE11) are more sensitive to topoisomerase damage than tdp1 mutants (Pouliot et al., 2001; Liu et al., 2002; Vance and Wilson, 2002). This suggests that activities other than Tdp1 act in parallel to repair topoisomerase I damage in vivo. It has been suggested that Tdp1 acts prior to the yeast phosphatases Apn1 and Apn2 in the Tdp1-repair pathway (Liu et al., 2002; Figure 4). Indeed, a triple mutant deficient in apn1 apn2 and the 3⬘-phosphatase gene TPP1 is hypersensitive to CPT. This sensitivity is relieved when the triple mutant becomes deficient in Tdp1 activity (Vance and Wilson, 2001). Repair of a cleavage complex necessarily involves removal of the peptide moiety. Conceptually, this can occur by specific cleavage (precise excision at the tyrosyl-phosphodiester bond) or nonspecifically by nucleolytic cleavage at an internal phosphodiester bond. The structure-specific endonucleases Rad1/ Rad10 and Mus81/Mms4 act at the boundary of 3⬘-ss/ ds-discontinuities on molecules containing flapped or branched DNA (see Figure 2B for substrates). Conceivably a cleavage complex could also be considered to possess a similarly distorted 3⬘-boundary. Growth tests in the presence of CPT reveal that RAD1, MUS81, and TDP1 act in nonoverlapping pathways to repair topoisomerase damage in S. cerevisiae (Liu et al., 2002; Vance and Wilson, 2002). However, a rad1 mus81 tdp1 triple mutant is still much less sensitive to the cytotoxic effects of CPT than a single mutant containing a defect in a gene involved in the RAD52 epistasis group (Liu et al., 2002). These observations suggest that previously uncharacterized/unassigned activities may act to repair topoisomerase damage in vivo (Figure 4). The existence of nonspecific pathways to repair topoisomerase I-DNA cleavage complexes raises the possibility that these systems can act on analogous lesions generated by similarly acting enzymes.

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Figure 4. Pathways for the Repair of Topoisomerase I Damage (A) Tdp1-mediated DSBR pathway. (B) Rad9mediated DSBR pathway. (C) Tdp1-mediated SSBR pathway (represented by broken arrows). Duplex DNA is shown in blue and covalently linked topoisomerase as a red circle. (1) Topoisomerase I-cleavage complex is formed from duplex DNA. (2) Nicked topoisomerase cleavage complex is converted to a DSB complex. (3) A signaling pathway mediated by histone H2AX, and probably MR, is activated. This stage has at least two alternative outcomes. In pathway (A), repair is mediated by a protease and Tdp1. In pathway (B), Rad9 is presumably involved in inducing the expression of genes (other than TDP1) that can act nucleolytically to process cleavage complexes. In pathway (A), it is possible the 26S proteosome (Desai et al., 2003; Xiao et al., 2003) acts to degrade the topoisomerase component of a cleavage complex. After the proteolytic step Tdp1 and a 3⬘-phosphatase (Tpp1, or Apn1, or Apn2 in yeast) act before the resulting DNA is fed into the DSBR pathway mediated by the RAD52 epistasis group, where again there may be a role for the MR complex. (4) A recombination intermediate is generated. (5) The recombination intermediate is presumably resolved to regenerate duplex DNA. In pathway (B), in yeast, signaling by Rad9 is followed by the action of nuclease complexes such as Rad1/Rad10 and Mus81/ Mms4, which operate in alternative pathways to each other and Tdp1. It is not clear if these enzymes act on structures prior to or subsequent to the formation of recombination intermediates (stage 4). Other nucleases may also exist that can act in parallel to Rad1/Rad10 and Mus81/Mms4. It is possible that the MR complex can act as such a nuclease. In pathway (C), at least in mammalian cells, Tdp1 operates in a SSBR pathway (see Caldecott, 2002, for details).

Pathways for the Repair of Type II TopoisomeraseCleavage Complexes As a 5⬘-tyrosyl-DNA phosphodiesterase (that could act specifically to remove a type II topoisomerase from a covalent complex) has never been identified, it is likely that the repair of these complexes is achieved mainly via nonspecific nucleolytic pathways. In contrast to type I topoisomerases, DNA replication, and other cellular events, such as replication fork-regression and endonuclease cleavage, appear to be required to convert a type II topoisomerase-cleavage complex into a cytotoxic lesion (see Hong and Kreuzer, 2003, and within). However, many of the gene products employed by cells to repair both types of topoisomerase-covalent complex are similar (Figure 5). This is suggested by the observation that cells deficient in genes involved in DSBR pathways are sensitive to agents that induce the formation of topoisomerase-I and-II cleavage complexes. In mammalian cell lines, mutations in genes involved in DSBR pathways also result in hypersensitivity to type II topoisomerase inhibitors (Jeggo et al., 1989; Caldecott et al., 1990). In S. cerevisiae, cells defective in Rad52 activity show increased sensitivity to the type II inhibitor

mAMSA (Nitiss and Wang, 1988). That recombinational processes are involved in repairing type II topoisomerase damage is suggested by the observation that homologous recombination is induced in eukaryotic cells treated with type II topoisomerase inhibitors (Nitiss and Wang, 1988; Pommier et al., 1988). Recent studies in S. cerevisiae indicate that type II topoisomerase-damage stimulates levels of homologous and nonhomologous recombination (Simon et al., 2000; Sabournin et al., 2003). Bacteriophage T4 has provided an excellent system to study the mechanisms of topoisomerase II-toxicity and repair (Kreuzer, 1998). In T4 it has been shown that a number of genes involved in recombinational repair are required to correct topoisomerase damage induced by the inhibitor mAMSA (Neece, et al., 1996; Woodworth, and Kreuzer, 1996; Stohr and Kreuzer, 2001). These include the uvsX and uvsY genes (analogous to the RAD51 and RAD52 paralogs of eukaryotes) whose products catalyze a DNA strand invasion reaction, the gp32 protein (functional equivalent of eukaryotic ssDNA binding protein RP-A), and the T4 Holliday junction resolving enzyme gp49 (Cromie et al., 2001). It is interesting to

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Figure 5. Pathways for the Repair of Topoisomerase II Damage (A) DSBR pathway mediated by homologous recombination proteins (see text). (B) DSBR pathways mediated by Ku70/Ku80 and/or Rad1/Rad10 (Sabournin et al., 2003). (C) Alternative pathway (represented by broken arrows) to formation of a cytotoxic lesion that is presumably repaired by DSBR pathways (Hong and Kreuzer, 2003). (D) It is possible the 26S proteosome (Desai et al., 2003; Xiao et al., 2003) acts to degrade the topoisomerase component of a cleavage complex. Alternative routes of repair are seen after proteasome action (represented by broken arrows). Duplex DNA is shown in blue and covalently linked topisomerase as a red circle. (1) Topoisomerase II-cleavage complex is formed from duplex DNA. (2) As S. cerevisiae Mre11/ Rad50 (T4 Gp46/47) is believed to act prior to end 5⬘-end resection we show these complexes acting on a topoisomerase II-cleavage complex prior to Holliday junction formation (see text). In Pathway A, the product generated is presumably a substrate for the single strand binding proteins Gp32 (RP-A). (3) At some stage the DSBR proteins Rad51/52/54 (T4 UvxX/Y) act to generate a recombination intermediate. (4) This structure is presumably resolved to regenerate duplex DNA.

note that, like S. cerevisiae, the T4 MR complex (gp46/ 47) is required to facilitate repair of a topoisomerase IIDNA covalent lesion (Neece, et al., 1996; Woodworth, and Kreuzer, 1996; Stohr and Kreuzer, 2001). Role of the Mre11/Rad50 Protein in Covalent Complex-Repair The Mre11/Rad50 (MR) complex mediates the repair of cleavage complexes; however, its role in this repair pathway remains controversial. MR complexes are found in organisms as widely diverged as bacteriophage T4 and humans (Sharples and Leach, 1995). MR complexes consist of a nuclease component known as Mre11, SbcD, or Gp46 (depending on the organism) and a structural, ATP binding, coiled-coil component known as Rad50, SbcC, or Gp47. Surprisingly, a strain of S. cerevisiae defective in MRE11 is more sensitive to damage induced by the topoisomerase I poison CPT than is a rad52 mutant (Liu et al., 2002). Rad50 mutants on the other hand are only as sensitive to CPT damage as rad52 mutants (Simon et al., 2000; Vance and Wilson, 2002). The higher sensitivity of the mre11 mutant suggests an involvement of the Mre11 nuclease in one of the steps in the pathway. However, it has been suggested that the nuclease function of the MR complex is not important for repairing topoisomerase I damage as a mutation that inactivates Mre11 nuclease function

confers little or no sensitivity to CPT (Liu et al., 2002, and within). This lack of sensitivity may derive from the activity of a nuclease that acts in an overlapping pathway to repair toposiomerase damage. Therefore, a careful study of mutants multiply deficient in these pathways is required. In Arabadopsis thaliana, D. melanogaster, S. cerevisiae, human, and mouse, the evolutionarily conserved Spo11 protein introduces DSBs into DNA during meiosis to initiate recombination (Keeney et al., 1997; Lichten, 2001). Several S. cerevisiae mutants have been isolated that contain point mutations in the RAD50 or MRE11 genes (rad50S and mre11S) (Alani et al., 1990). In these strains, the Spo11 molecule remains covalently attached to DNA at the site of the break (Keeney et al., 1997). In this instance the Spo11-DNA cleavage complex does not arise through the action of a drug. It has been proposed that, as the MR complex functions as a nuclease implicated in a remarkable variety of reactions involving ds-ends (D’Amours and Jackson, 2002; Connelly et al., 2003), it can act directly on the Spo11-covalent complex to catalyze its removal (Keeney et al., 1997). However, MR complexes can act as DNA damage sensors; it is possible therefore that they also (or alternatively) play an important role in detecting and signaling the presence of cleavage complexes to checkpoint proteins (D’Amours and Jackson, 2001; Usui et al., 2001).

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MR complexes have been shown to mediate the removal of a covalently attached viral protein from a DNA terminus. To circumvent the “end-replication problem” (Watson, 1972) and protect their DNA termini, many viruses covalently attach terminal-proteins to their genomes. These protein-DNA structures are analogous to the cleavage complexes formed by DNA processing enzymes that act via a transient covalent intermediate. The dsDNA virus, adenovirus, has a terminal protein (pTP) attached to its termini that must be removed before viral concatemerization can occur. When examining what host factors were required for concatamerization it was discovered that Mre11 was essential. Furthermore, this concatamerization did not occur in a cell line that was mutated in a Mre11 motif associated solely with nuclease activity (i.e., these mutants are wild-type in other aspects of Mre11 function). It was suggested that the nuclease (not the signaling) function of Mre11 is required for viral end-joining and that the human MR complex might be acting directly to remove pTP from the viral genome (Stracker, et al., 2002). In this instance, the MR complex is, once again, mediating the removal of a naturally occurring covalent complex from a DNA molecule. In vivo, the nucleolytic activities of MR complexes are associated with a number of DNA-terminal deprotecting roles. For example, they are believed to be responsible for unblocking DNA ends sealed by hairpin structures (D’Amours and Jackson, 2002; Connelly et al., 2003). Recently, Connelly et al. (2003) have provided in vitro evidence that another MR complex, E. coli SbcCD, can act nucleolytically to process a DNA molecule covalently bound to biotin and complexed with streptavidin. When SbcCD was incubated, under conditions where the nuclease activity of the enzyme was restricted to the termini of a DNA duplex, it removed the biotin-streptavidin complex from a DNA end by introducing a doublestrand break. This observation indicates that a MR complex has the potential to act adjacent to a protein molecule linked to a DNA terminus in a nucleolytic fashion. How a MR complex might act in a cleavage complex repair pathway is suggested in Figures 4 and 5.

earmarked for degradation and the putative proteases involved also remains to be determined. Recent clues suggest that transcriptional stalling and modification of topoisomerases by ubiquitin-like proteins may single them out as being candidates for degradation by a cellular proteasome (Horie et al., 2002; Desai et al., 2003; Xiao et al., 2003). Investigating how cleavage complex repair is integrated with existing DNA repair and-checkpoint systems will provide fascinating insights into the hierarchical organization of such pathways in vivo. As we begin to understand these complexities, the hope is that we will also begin to understand how to design better treatments for cancers susceptible to the stabilization of protein-DNA covalent complexes.

Conclusions Proteins covalently linked to DNA pose a threat to genome integrity and cell viability. A number of enzymes have the potential to remain covalently bound to DNA; the best-characterized examples are the type I and type II topoisomerases whose cleavage complexes are trapped by antibiotic and chemotherapeutic drugs. The effectiveness of these drugs is modulated by the pathways employed to repair this type of damage. The enzyme Tdp1 is used to repair a specific protein-linked lesion but substantial redundancy exists in the pathways that deal with cleavage complex-damage in vivo. Several nonspecific, nucleolytic, pathways for the repair of cleavage complexes exist, and many proteins involved in SSBR-and DSBR-processes are implicated in them. Among these the MR complex plays a key role in facilitating repair but the function of its nuclease activity in this process remains controversial. How a DNA molecule covalently linked to protein is differentiated from a noncovalently linked protein-DNA complex is unclear. The way in which these proteins are

D’Amours, D., and Jackson, S.P. (2001). The yeast Xrs2 complex functions in S phase checkpoint regulations. Genes Dev. 15, 2238– 2249.

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