The topological state of DNA in cells is regulated by the action of DNA topoisomerases (Bush et al., 2015; Corbett and Berger, 2004; Pommier et al., 2016; Wang, 2002). Different cellular process, such as transcription and recombination, can alter the topology of DNA and the action of topoisomerases helps maintain the correct topological state. In order to change the topology of DNA, topoisomerases transiently break either one or two strands of DNA in order to allow movement of strands before the breaks are resealed. In this manner, topoisomerases can relax and supercoil DNA, catenate/decatenate, and knot/unknot DNA molecules, and in some cases RNA as well (Ahmad et al., 2016; Wang et al., 1996). Due to their involvement in crucial cellular processes, topoisomerases are the target of important chemotherapeutic agents (Pommier et al., 2010).

Topoisomerases are found in all three domains of life, but the diversity in structure and sequence suggests that the major subtypes evolved independently (Forterre et al., 2007). All topoisomerases have in common the use of phosphotyrosine intermediates for transient cleavage through a transesterification mechanism (Corbett and Berger, 2004). Topoisomerases are classified into two types (I and II) based on whether they cleave one or two strands of DNA in a concerted manner. Topoisomerases are subclassified based on similarities at the sequence and structural levels. Type I enzymes, which cleave one DNA strand, are subclassified into three subtypes, IA, IB, and IC. Type IA enzymes are found in bacteria, archaea, and eukarya, employ an enzyme-bridged strand passage mechanism, and share a strand passage structural domain with a very characteristic toroidal shape (Lima et al., 1994). Type IB enzymes are also found in all domains of life, employ a swiveling or controlled rotation mechanism, and show clear structural and sequence similarities, despite some of them being much larger than other members of the same subtype (Corbett and Berger, 2004). The structure and mechanism of type IA and IB topoisomerases and their cellular role are well studied, and in both subtypes the general steps of the DNA-binding and cleavage/religation mechanism are well understood.

Topoisomerases of the third subtype, IC, have only been found in the archaeal Methanopyrus genus (Forterre, 2006) and relax DNA using a controlled rotation/swiveling mechanism (Taneja et al., 2007). Its only member is topoisomerase V, which was originally described as a type IB enzyme (Slesarev et al., 1993), but later classified into its own subtype due to the lack of sequence or structural similarities with other topoisomerases (Forterre, 2006; Taneja et al., 2006). The exact cellular role of topoisomerase V in the hyperthermophile Methanopyrus kandleri is not known. Topoisomerase V works not only at extremely high temperatures (65–122°C), but also in high salt concentrations (Slesarev et al., 1994). It is also an unusual protein as it combines DNA repair and topoisomerization activities in the same polypeptide (Belova et al., 2001; Rajan et al., 2016; Rajan et al., 2013), a unique feature of this enzyme. Topoisomerase V is formed by a small, ~30-kDa topoisomerase domain followed by 24 tandem helix–hairpin–helix (HhH) repeats (Doherty et al., 1996) arranged as 12 (HhH)₂ domains (Belova et al., 2002; Shao and Grishin, 2000). HhH repeats are associated with DNA lyase repair activity (Doherty et al., 1996; Shao and Grishin, 2000) and 3 of the 12 (HhH)₂ domains have apurinic/apyrimidinic (AP) lyase and deoxyribose-5-phosphate (dRP) lyase activities (Rajan et al., 2016; Rajan et al., 2013). These 12 (HhH)₂ domains also stimulate the topoisomerase activity as mutant topoisomerase V proteins with fewer repeats show decreasing relaxation activity (Belova et al., 2002; Rajan et al., 2010). (HhH)₂ domains are usually found as isolated domains, not in tandem arrangements (Shao and Grishin, 2000), which is another unusual characteristic of topoisomerase V. The topoisomerase domain has no sequence or structural similarities to other topoisomerases or any other protein, making it a unique fold (Forterre, 2006; Taneja et al., 2006). The active site residues have been identified (Rajan et al., 2014), and their three dimensional arrangement suggests a different catalytic mechanism from other topoisomerases (Rajan et al., 2014). In the structures of the free protein, the topoisomerase domain active site is inaccessible as it is covered by the (HhH)₂ domains (Rajan et al., 2016; Rajan et al., 2013; Rajan et al., 2010; Taneja et al., 2006).

The way that topoisomerase V interacts with DNA, relaxes it, and recognizes lesions is not known. This stands in contrast to all other topoisomerase types, either I or II, where structures of complexes of the proteins with DNA are known (Bush et al., 2015; Corbett and Berger, 2004). The absence of structural information on topoisomerase V in complex with DNA has hampered efforts to understand fully not only its mechanism of DNA relaxation, but also the way AP/dRP lyase and topoisomerase activities are coupled and the conformational changes needed to expose the active site and bind DNA. Here, we present structures of topoisomerase V in complex with DNA containing an abasic site. The structures show the way the (HhH)₂ domains embrace DNA and change conformation to expose the topoisomerase active site and accept DNA into it. Exposure of the active site involves breaking of a long helix linking the topoisomerase and (HhH)₂ domains. The novel manner employed by topoisomerase V to bind DNA suggests that the (HhH)₂ domains serve as a processivity factor in addition to containing the repair active sites. Based on the structural findings, important regions were identified and site-directed mutagenesis studies probed the role of different residues in DNA relaxation. Despite type IB and IC enzymes both using a controlled rotation DNA relaxation mechanism (Koster et al., 2005; Taneja et al., 2007), the structures show that there are significant and substantial differences; topoisomerase V bends DNA sharply at the active site resulting in melting of the DNA, which may facilitate strand rotation. The structures establish that type IC enzymes share no features with other type of topoisomerases at any level. They form a distinct subtype employing a different mechanism of DNA cleavage/religation and relaxation. The structures not only bring our understanding of type IC enzymes to a similar level as for all other topoisomerases, but also demonstrate a different mechanism of DNA relaxation that has not been observed before. Topoisomerase V, due to its unusual characteristics, continues to expand our understanding of mechanisms of DNA topological manipulations and exemplifies a solution to the way proteins relax DNA that is different from the ones employed by other topoisomerases.

The general outlines of the DNA-binding mode and relaxation mechanism is understood for many topoisomerases (Bush et al., 2015; Corbett and Berger, 2004), but this is not the case for type IC enzymes. In the latter case, the absence of information on the way they recognize and bind DNA has limited our understanding of the mechanism of this topoisomerase subtype. In addition, the lack of sequence or structural similarities between topoisomerase V and other topoisomerases meant that it was not possible to deduce the DNA-binding mechanism based only on similarities. The presence of multiple (HhH)₂ domains as well as biochemical information suggested that these domains were involved in DNA binding (Belova et al., 2002; Rajan et al., 2010), but it was not clear how tandem domains recognize and bind DNA. The structures presented here show the way topoisomerase V interacts with DNA, both through the (HhH)₂ domains and the topoisomerase domain. DNA binding by the (HhH)₂ domains is unusual and, in a way, not previously observed for other proteins. The tandem (HhH)₂ domains wrap around the DNA forming a loop-like structure that encircles the DNA. Typically, HhH repeats are found as single repeats or forming (HhH)₂ domains (Shao and Grishin, 2000), not as tandem arrangements. Some proteins that include (HhH)₂ domains dimerize to have two (HhH)₂ domains next to each other, for example XPF endonucleases (Nowotny and Gaur, 2016), but the (HhH)₂ domains are not arranged in a tandem configuration in the protein. For this reason, even though HhH repeats and (HhH)₂ domains are well studied, it was not possible to model DNA binding by topoisomerase V based on known structures. Unlike other (HhH)₂ domain-containing proteins, where the domains are involved in DNA binding and repair, in topoisomerase V some (HhH)₂ domains surround the DNA but do not contact it, others contact it, and only three out of twelve are directly involved in repair. When comparing individual (HhH)₂ domains with repair enzymes that interact with DNA the interaction observed in topoisomerase V is different from the one observed in other (HhH)₂ domain complexes with DNA. It is not possible to establish whether the topoisomerase V repeats directly involved in DNA repair bind DNA similar to the way other (HhH)₂ domain-containing DNA repair enzymes do. The (HhH)₂ domains that contain the AP/dRP lyase activity do not engage directly with the DNA abasic sites in the structure. For this reason, the question on how topoisomerase V recognizes DNA lesions is not completely answered by the current structure. To understand the way the enzyme recognizes and cleaves abasic sites additional structural information on the interactions of the repair domains and an abasic site is needed.

The overall DNA-binding mode by topoisomerase V is unusual. The (HhH)₂ domains surround the DNA loosely covering almost four helical turns. Topoisomerase V clamps around DNA by having two sets of tandem repeats of (HhH)₂ domains that follow the path of DNA in opposite directions. The region between repeats 7 and 8 serves as the turning point to permit the repeats to change direction and encircle the DNA. The Topo-97(ΔRS2) fragment is missing the last two (HhH)₂ domains plus a few amino acids of unknown structure at the C-terminus. Based on the structures and the modeling studies described, it is likely that the last two (HhH)₂ domains continue the same path as the previous three and interact with the topoisomerase domain and completely enclose the DNA forming two full loops (Figure 2—figure supplement 4). Given the length of DNA covered, there are few direct interactions with the phosphate backbone and very few with the nucleobases. Instead, it appears that the (HhH)₂ domains create an enclosed positively charged track or groove where the DNA can travel. Other proteins that use tandem repeats of DNA-binding domains do not make a turn to change direction while binding DNA. Instead, they wrap around the DNA in a single direction. For example, zinc finger containing proteins such as Zif268 or Gli (Pavletich and Pabo, 1991; Pavletich and Pabo, 1993) and TAL effector proteins binding domains (Mak et al., 2012) interact with the DNA by having tandem repeats that wrap around the DNA, but they extend along the DNA, not turning to form a loop-like structure that surrounds it.

DNA binding by topoisomerase V does not require all (HhH)₂ domains, but its activity is enhanced when more (HhH)₂ domains are present, suggesting that even a few domains are capable of binding DNA (Belova et al., 2002; Rajan et al., 2010). Surrounding DNA is not necessary for activity, but it clearly enhances it. Furthermore, it has been shown that the topoisomerase V (HhH)₂ domains can be used to enhance processivity of other enzymes (Pavlov et al., 2002). Thus, the (HhH)₂ domains may serve a similar role as sliding clamps that surround DNA (Hedglin et al., 2013), which enhance processivity by keeping polymerases closely associated with DNA while allowing movement along the DNA. Unlike sliding clamps, which form a ring around DNA and need a clamp loader to assemble, the topoisomerase V (HhH)₂ domains surround the DNA in a more extended manner and load into DNA without assistance.

The active site of topoisomerase V had not been observed in an accessible conformation before. All other known structures (Rajan et al., 2016; Rajan et al., 2013; Rajan et al., 2010; Taneja et al., 2006), aside from the structure of the isolated topoisomerase domain (Rajan et al., 2010), showed the active site obstructed by the (HhH)₂ domains. Whereas it was clear that the (HhH)₂ domains had to move to allow access to the active site, it was not clear how this is accomplished. The structure of the symmetric complex with DNA shows that the topoisomerase domain moves relative to the (HhH)₂ domains through a single conformational change, a kink in the helix linking the first (HhH)₂ domain and the topoisomerase domain. The domains themselves move as rigid bodies; the only change occurs in the linker helix. This simple mechanism allows the domains to separate and exposes the active site. In addition, the linker helix interacts with the DNA directly, suggesting that the conformational change could be triggered by protein DNA interactions. Single mutations in the linker helix did not disrupt activity, aside from one, Leu290Pro. Arginines 288 and 293 face the major groove and single mutations to alanine had no effect. A double mutant with both arginines changed to alanines had reduced activity whereas reversing the charge of these residues, Arg288Glu/Arg293Glu, completely abolished activity. This suggest that the overall charge character of the region is important for protein/DNA interactions as the positively charge side chains may facilitate the approach of the DNA to the linker helix. Leu290Pro was designed to disrupt the helical structure of the linker helix. It is positioned at the end of the break point in the linker helix. The presence of the proline abolished activity, supporting the notion that the structure and plasticity of the linker helix is very important for activity. One possible explanation for the loss of activity is that the proline prevents the formation of the linker helix in the closed or DNA-free form, thus affecting the ability of the protein to recognize and bind DNA and attain the proper conformation that allows binding of the DNA by the (HhH)₂ domains and orienting the topoisomerase domain properly for catalytic activity.

The movement of the domains also exposes a large, positively charged patch in the topoisomerase domain where the DNA enters the active site. In order to enter the active site, the DNA bends at two positions. There is small bend adjacent to the abasic site and a much larger bend where the DNA enters the topoisomerase domain. It is unlikely that the abasic site-induced bending reflects a feature of DNA binding and recognition as nondamaged DNA is easily relaxed by topoisomerase V. Surprisingly, the DNA is highly bent where it enters the topoisomerase domain and the bending has an unusual consequence, the double stranded DNA melts to allow only one strand to enter the active site. The two DNA strands separate, breaking the base pairing between them. One strand, the noncleaved one, is anchored to the protein by clamping on a nucleobase. The other strand enters the active site and approaches the active site tyrosine. Both DNA strands exit the protein almost parallel to each other, suggesting that base pairing would resume once the DNA exits the protein. Single mutations of positively charged amino acids leading to the positively charged patch have, in general, a minor effect. Arg37, Lys47, His56, Arg134, and Arg135 were mutated to alanine with almost no reduction in relaxation activity. Interestingly, a double mutant where Lys134 and Arg135 were mutated to alanines had minimal activity, but when these amino acids were mutated to glutamate, reversing the charge of the region, activity was completely lost. These results suggest that single changes do not alter enough of the charge character of the region to significantly reduce the activity, but that more extensive changes do affect the activity and, finally, that the positively charge character of the region is crucial for activity. The mutation Ala132Ile was designed to probe the structure of a region that is in intimate contact with the cleaved DNA strand through protein backbone interactions. This mutant showed significant loss of activity, even though the side chain does not face the DNA. Ala132 faces the interior of the protein and it is likely that the bulkier isoleucine side chain affected the structure of the region and hindered protein/DNA interactions, supporting the notion that the structure of this region is important for protein/DNA interactions. Mutations of charged amino acids in contact with the noncleaved DNA strand had a larger effect on activity. Arg109Ala did not show any change, but Arg83Ala and Arg108Ala show significant decrease in activity. In particular Arg108Ala abolishes activity completely. Their position in the complex suggests that Arg83 and Arg108 may interact with the noncleaved strand and hold it in place during DNA relaxation activity. It is interesting to note that Arg108Ala shows no activity and is near the active site, suggesting that this amino acid may also play a role in catalysis that was not noticed before. Not surprising, the double mutant of Arg108 and Arg109 to alanines show no activity.

The residues forming the topoisomerase active site were recognized based on structural and biochemical observations (Rajan et al., 2014; Taneja et al., 2006). The complex structure confirms that Arg131, Arg144, His200, Glu215, Lys218, and Tyr226 are involved in interacting with DNA and helps understand their role in the DNA cleavage/religation reaction. The two arginines, Arg131 and Arg144, interact directly with the phosphate group adjacent to the scissile bond (P-1) and are likely to interact also with the scissile bond phosphate (P0). Arg144 also forms a hydrogen bond to the OH group of the active site tyrosine. Mutations in either arginine led to very reduced activity (Rajan et al., 2014), suggesting an important role for these residues in the reaction. It was suggested (Rajan et al., 2014) that these arginines play a role in transition state stabilization, which would be broadly consistent with the observations from the complex structure. Glu215, an unusual residue due to its negative charge, comes close to the phosphate backbone and hydrogen bonds to His200. It was observed before that Glu215 reduces the binding affinity of the protein for DNA, which would be consistent with a negatively charged residue approaching the negatively charged phosphate backbone (Rajan et al., 2014). In the complex structure, His200 does not seem to interact directly with DNA so its role in the cleavage/religation reaction is not clear. Finally, the role of Lys218 is also not clear from the structure, as it is not observed in a position to interact with DNA, probably due to the absence of P0. It appears that it could interact with the phosphotyrosine intermediate helping stabilize it after cleavage; mutational studies show that the lysine is essential (Rajan et al., 2014). Finally, the finding that Arg108 is essential for activity suggests that it may play a role in catalysis, either through a direct role in cleavage/religation or stabilizing the DNA, that had not been recognized before. The combined structural and biochemical studies do suggest that the mechanism of cleavage and religation is different from the one employed by type IB enzymes. In type IB enzymes the histidine in the catalytic pentad interacts directly with DNA and the lysine plays a role in proton transfer. This does not appear to be the case in type IC topoisomerases. The presence of the glutamate in close contact with the phosphate backbone as well as the potential different role of His200 and Lys218 suggest that a distinct catalytic strategy is employed by topoisomerase V. Additional structural studies to capture covalent intermediates are needed to understand the cleavage/religation reaction.

Both type IB and IC topoisomerases use a swiveling mechanism to relax DNA. Structures of type IB enzymes in complex with DNA (Perry et al., 2006; Redinbo et al., 1998) show that the DNA remains double helical and largely unbent and is surrounded by the protein (Figure 6). Cleavage and formation of a transient phosphotyrosine intermediate allow the intact strand to rotate until the broken free DNA strand is recaptured and ligated. Interaction of the DNA with the protein causes friction, which modulates the reaction (Koster et al., 2005). Single molecule experiments on topoisomerase V show a similar overall mechanism where friction also plays a role (Taneja et al., 2007), suggesting that type IB and IC enzymes employ the same general strategy. Given these parallel strategies, the presence of a sharp bend and a single stranded region in the complex of topoisomerase V was unexpected as type IB topoisomerases do not bend or melt the DNA (Perry et al., 2006; Redinbo et al., 1998). Single stranded DNA recognition and binding is part of the type IA mechanism, but these enzymes work by an enzyme-bridged strand passage mechanism, which is fundamentally different from the swiveling mechanism employed by type IB and IC enzymes. These observations suggest that despite the apparent similarities, type IC enzymes employ a different relaxation strategy. Unlike type IB enzymes, type IC molecules bend the DNA to create a single stranded region that is likely to facilitate swiveling by freeing the two DNA strands around the cleavage site. It is not clear whether the rotation of the strands involves only rotation of the strands or also movement of the topoisomerase domain; it is possible that the topoisomerase domain moves as the strands rotate (Figure 6A). Similar to type IB enzymes, the broken strand is captured after swiveling around the other strand. Finally, it is interesting to note that whereas type IB enzymes surround the DNA during the reaction, type IC enzymes do not appear to do so. The (HhH)₂ domains surround the DNA, but their role seems to be to act as a processivity factor as these domains are not required for DNA relaxation (Rajan et al., 2010). Thus, while there are some overall similarities between the two subtypes, there are important differences that suggest that topoisomerase V employs a completely different relaxation mechanism.

Figure 6

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It has been observed previously that type IB enzymes bind at crossover points in supercoiled DNA and that this may provide a mechanism for overall topology sensing (Madden et al., 1995; Zechiedrich and Osheroff, 1990). Two independent DNA-binding sites in the protein are needed to bind two strands of DNA at a crossover point. Crossover binding provides a facile way to recognize the overall topology of DNA as, in general, only supercoiled DNA, either positively or negatively supercoiled, will form crossovers. Viral type IB topoisomerases have been observed to synapse DNA strands (Moreno-Herrero et al., 2005; Shuman et al., 1997), again suggesting the presence of more than one DNA-binding site. Whereas there are no structures of any topoisomerase binding two DNA strands simultaneously, there are electron (Shuman et al., 1997; Zechiedrich and Osheroff, 1990) and atomic force microscopy (Moreno-Herrero et al., 2005) studies that support the notion of multiple binding sites as well as a structure of a bacterial type IB enzyme bound to a secondary DNA site (Patel et al., 2010). The latter structure supports the notion that type IB enzymes can bind two DNA molecules simultaneously and independently. It is possible that topoisomerase V, as has been suggested for type IB enzymes, recognizes the overall topology of DNA by preferentially binding at crossover points. The structure of the asymmetric complex of topoisomerase V with DNA shows the presence of a second DNA-binding site that involves the external surface of the (HhH)₂ domains and that does not interfere with binding of the strand that enters the active site (Figure 1). Figure 6B shows a cartoon of topoisomerase V bound to two DNA segments. It shows that it could be possible for topoisomerase V to recognize the overall DNA topology by simultaneously binding two DNA strands. This would make type IC topoisomerases share with type IB enzymes not only a similar relaxation mechanism, controlled strand rotation, but possibly an overall topology sensing mechanism. Whether type IB and/or IC enzymes use a crossover point binding mechanism to sense overall topology remains to be proven, but it adds to the mechanistic similarities between the two types of topoisomerases despite their very different structures and supports the notion of convergent evolution to a common mechanism of DNA topology sensing and relaxation.

The structures presented here add significantly to our understanding of type IC enzymes and their mechanism. The structures show that an important role of the (HhH)₂ domains is to surround DNA and, in this manner, serve as a processivity factor. In this way, they keep the enzyme bound to DNA, probably allowing it to travel along DNA. It is not clear from the structures how DNA lesions are recognized, but if the protein slides along the DNA it could scan it for lesions using the (HhH)₂ domains containing the repair active sites. The topoisomerase domain is likely to be obstructed in the absence of DNA/protein interactions, but a simple conformational change around a linker helix exposes the active site once the protein is bound to DNA. The DNA bends tightly as it interacts with the protein around the active site region, leading to single stranded DNA formation, which may help facilitate strand rotation. Finally, the catalytic mechanism of DNA cleavage and religation appears to be different from the one employed by other topoisomerases, despite some very general similarities. All these observations indicate that topoisomerase V is a multifaceted enzyme that encompasses in the same polypeptide a novel topoisomerase domain, DNA repair domains, and a DNA clamp. Thus, type IC enzymes are fundamentally different from all other topoisomerases. The existing structural, biochemical, and biophysical data help to establish firmly type IC topoisomerases as a completely different topoisomerase subtype with few sequence, structural, or mechanistic similarities to all the other subtypes.

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under the accession numbers 8DF7, 8DF8, 8DF9, and 8DFB.

1. 1. Mondragón A
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Author details

1. Amy Osterman

   Department of Molecular Biosciences, Northwestern University, Evanston, United States

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

2. Alfonso Mondragón

   Department of Molecular Biosciences, Northwestern University, Evanston, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   a-mondragon@northwestern.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0423-6323

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