All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, Eukaryotic topoisomerase I (topo I)1 is a monomeric enzyme that plays a major role in important cellular processes by regulating the topology of DNA. The enzyme relaxes negative and positive supercoils arising as a consequence of DNA processes such as DNA transcription, replication, recombination, and chromosome condensation (1). Mechanistically, topo I acts by introducing transient singlestrand breaks into the DNA double helix. The catalytic cycle can be subdivided into several steps including: (i) non-covalent DNA binding, (ii) cleavage, (iii) strand rotation, (iv) religation, and (v) enzyme turnover. The cleavage and religation events constitute two reverse phosphoryl transfer (transesterification) reactions. During the cleavage reaction, an active-site tyrosine residue of the enzyme is used as a nucleophile to break a phosphodiester bond of the DNA backbone, generating a covalent enzyme-(3Ј-phosphotyrosyl)-DNA linkage and a free 5Ј-hydroxyl group (2-4). This 5Ј-hydroxyl group provides the nucleophile for the religation reaction that restores intact DNA.The solved crystal structure of an N-terminal-truncated version of the human topo I together with proteolytic analyses show that the enzyme is organized into four structural domains. These consist of an N-terminal domain (amino acids 1-206), a core domain (amino acids 207-635), a linker domain (amino acids 636 -712), and a C-terminal domain (amino acids 713-765) (2, 3). The C-terminal domain contains the active-site tyrosine (Tyr 723 ), which together with the catalytic residues Arg 488 , Lys 532 , Arg 590 , and His 632 of the core domain constitutes the active site of the enzyme (4 -8). Structural data show that the core and C-terminal domains form a clamp structure that wraps around the DNA and, together with the helix-turnhelix linker domain (1), contacts DNA in a region extending 4 base pairs upstream and 9 base pairs downstream of the cleavage site (3). Based on this structural information of the human topo I-DNA complex, a model for strand rotation (topoisomerization) has been proposed. According to this "controlled rotation" model, rotation of the cleaved strand around the intact strand is partially hindered by contacts between the rotating DNA and part of the core and linker domains (3). The involvement of the linker ...

DNA topoisomerase II is a multifunctional and highly complex enzyme that is able to change the topological conformation of DNA in response to different physiological alterations (1-3). Topoisomerase II enzymes mediate topological changes by introducing a transient break in one DNA duplex, the G segment, while another duplex, the T segment, is coordinately transported through the gated DNA. During the process, ATP is required to drive the enzyme through a series of dramatic conformational changes dependent on both interdomain and intersubunit communication (4 -8).Eukaryotic topoisomerase II is a homodimeric enzyme consisting of three distinct regions. The N-terminal and core regions, containing the two catalytic entities, are highly conserved among eukaryotic organisms and also share homology with DNA gyrase, which represents the bacterial DNA topoisomerase II counterpart. The active site for ATP hydrolysis is encompassed in the N-terminal region, whereas that of DNA cleavage and ligation is located in the central region. The C-terminal part shows no sequence conservation and is dispensable for catalytic activity in vitro (9 -11).The N-terminal region of topoisomerase II forms an ATPoperated clamp that closes upon ATP binding, allowing trapping of the T segment (6,8,12). The crystal structures of the yeast and bacterial N-terminal topoisomerase II fragments reveal that the dimeric N-terminal clamp contains two domains in each protomer (8,12). The most N-terminal domain holds the ATP-binding site that dimerizes upon nucleotide binding (8,12,13). This domain shares homology with the GHKL-type ATPases including topoVI-B (14) and MutL (15), for which a similar clamp operation has been observed. The second domain, called the transducer domain, bridges the N-terminal ATPase domain and the core region. Based on the yeast topoisomerase II structure, the transducer domain comprises the walls in a 6-Å-wide hole formed upon clamp closure, and it has been suggested to push the T segment through the DNA gate (12). The domain furthermore contains a loop that extends into the ATP-binding pocket and harbors a highly conserved lysine that contacts the ␥-phosphate of the bound nucleotide (8,12,13). Based on structural and biochemical analyses of the loop region, communication between the ATP-binding GHKL domain and the central domain of the enzyme responsible for DNA cleavage/ligation has been suggested to go through the transducer domain (13, 16). Upon nucleotide binding and closure of the N-terminal clamp, the transducer domain undergoes a large domain rotation, probably facilitating opening of the DNA gate in the G segment bound by the central domain (14). Recent studies surprisingly revealed that the transducer domain also bears structural homology to MutL and the archea topoVI-B, and similar conformational effects of nucleotide binding have been reported for the transducer domain of these GHKL-ATPases, indicating that the structure and motion of this domain play a conserved role in the clamp mechanism (14).According to the av...

Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra-and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II␣ heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional Nterminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II␣ can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.DNA topoisomerase II is a multifunctional and highly complex enzyme, which uses the energy of ATP to resolve topological problems generated during DNA metabolic processes, including DNA replication, transcription, and recombination (1, 2). Beyond these functions, topoisomerase II is an abundant component of the mitotic chromosome scaffold (3), and it alleviates constraints in the DNA during chromosome segregation and condensation (1).To preserve the topological integrity of the genome, the dimeric topoisomerase II enzyme strictly controls the passage of one duplex DNA (the T-segment) through another duplex (the G-segment) coordinately cleaved by the enzyme. ATP is required during the process to drive the enzyme through a series of conformations, which are essential to topoisomerase II catalysis.During the last years, extensive research has provided valuable information about the usage of ATP during the reaction pathway of topoisomerase II, but still several steps in this highly complex process remain to be unraveled. A study of the ATP consumption in yeast topoisomerase II has indicated that a tight coupling exists between ATP utilization and DNA transport under unsaturated ATP concentrations (5). However, a human topoisomerase II enzyme lacking amino acids 350 -407 at the interface between the ATPase and the cleavage/ligation domain was unable to perform strand passage, although ATPase and cleavage activities were intact (6). These results indicate that correct interdomain communication as well as signaling of ATP binding and hydrolysis to the rest of the enzyme are essential for the coupling of ATP consumption and DNA transport.The binding of ATP is cooperative in the presence of DNA (5) and ...

We have characterized a human topoisomerase IIalpha enzyme with a deletion of the conserved QTK loop, which extends from the transducer domain to the ATP-binding pocket in the GHKL domain. The loop has been suggested to play a role for interdomain communication in type II topoisomerases. The mutant enzyme performs only very low levels of strand passage, although it is able to cleave and ligate DNA as well as close the N-terminal clamp. Cleavage is nearly unaffected by ATP and ATP analogues relative to the wild-type enzyme. Although the enzyme is able to close the clamp, the clamp has altered characteristics, allowing trapping of DNA also in the absence of an ATP analogue. The enzyme furthermore retains intrinsic levels of ATPase activity, but the activity is not stimulated by DNA. Our observations demonstrate that the QTK loop is an important player for the interdomain communication in human topoisomerase IIalpha. First, the loop seems to play a role in keeping the N-terminal clamp in an open conformation when no nucleotide is present. Once the nucleotide binds, it facilitates clamp closure, although it is not essential for this event. The QTK loop, in contrast, is essential for the DNA-stimulated ATPase activity of human topoisomerase IIalpha.

Members of the RecQ helicase family are mutated in several human genomic instability syndromes, such as Werner and Bloom syndromes. The syndromes are characterized by premature ageing and cancer predisposition, respectively, and are therefore extensively used as model systems for studies of ageing and cancer. RecQ homologues are widely expressed enzymes, and genetic and biochemical investigations have pointed to their involvement in homologous recombinational DNA repair pathways. In the review we will focus on the implications of RecQ helicases for genome maintenance with specific emphasis on the homologues found in yeast.