The Transducer Domain Is Important for Clamp Operation in Human DNA Topoisomerase IIα

Abstract

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...

Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

“…Based on the newly published structure of the N-terminal fragment of yeast topoisomerase II (10), we find it very likely that the insertion impairs the strand passage reaction via sterical hindrance. Our results show that the insertion at position 351 slightly increases topoisomerase II-mediated DNA cleavage complex formation, consistent with a role of the transducer domain in controlling DNA cleavage (15). The biochemical characterization of 351i has revealed further that the enzyme retains DNA-stimulated ATP hydrolysis, although at a lower level compared with that of the wild-type enzyme.…”

“…The DNA cleavage level displayed by 351i in the absence of nucleotide slightly exceeds that of the wild-type enzyme. This catalytic trait is shared with the enzyme bearing an insertion at position 408 in the transducer domain (15) and indicates that an important function of the transducer domain is to control the cleavage level of the core domain.…”

“…However, a coordination is required to obtain topoisomerase activity (4). Communication between the ATPase domain and the central domain of the enzyme responsible for DNA cleavage/ligation seems to go through the transducer domain (4,(13)(14)(15). The conformational changes caused by ATP binding to the N-terminal region thus reach all the way to the core domain and also change the catalytic features of the DNA cleavage/ligation reaction of the enzyme.…”

“…We previously characterized a human topoisomerase II␣ enzyme bearing an insertion at position 408 in the transducer domain (15), which based on alignment with the crystallized N-terminal fragment from yeast topoisomerase II is located close to position 351 in the tertiary structure. However, the two insertions result in enzymes with very different phenotypes where only 351i is able to perform clamp closure.…”

“…The second domain, called the transducer domain, forms the walls of the clamp and connects it to the enzyme core. Furthermore, communication between the ATP-binding domain and the central domain of the enzyme, responsible for DNA cleavage/ligation, has been suggested to go through the transducer domain (4,(12)(13)(14)(15). Upon clamp closure, the transducer domain of each protomer approaches one another, creating a very tight cavity that is actually too small to hold a T-segment (10).…”