Use this URL to cite or link to this record in EThOS:
Title: The ATP-operated clamp of human DNA topoisomerase IIα
Author: Campbell, Spencer
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2001
Availability of Full Text:
Access from EThOS:
Access from Institution:
The type II DNA topoisomerases operate via a complex multistep mechanism: one segment of DNA (the T segment) is captured by the N-terminal domains and directed through a transient break in another DNA segment held by the core of the enzyme (the G segment). In most reactions of type II enzymes, this process is dependent upon the binding and hydrolysis of ATP by the N-terminal domains. I have examined the catalytic properties of the N-terminal ATPase domain of human DNA topoisomerase Ila. The intrinsic ATPase activity of this N-terminal domain is stimulated by DNA in a length-dependent manner. A DNA fragment of 10 base pairs is sufficient to stimulate the ATPase activity of this enzyme. With pBR322 as the DNA cofactor, the ATPase activity is hyperstimulated at a base pair to enzyme dimer ratio of ~50:1. With short DNA fragments of 28-44 base pairs, the hyperstimulation of the ATPase activity is maximal with a 38 base pair DNA fragment. This hyperstimulation phenomenon was previously attributed to interactions between adjacent enzyme dimers on a DNA strand, and we have interpreted our results in the light of this model. The physiological significance of this hyperstimulated ATPase activity is not yet known. The ability of the N-terminal domain to bind DNA and to dimerise in the presence and absence of nucleotide has been investigated. The enzyme is a monomer in solution, but in the presence of ADPNP a large conformational shift occurs, such that the enzyme associates into a dimer and a higher order species, possibly a tetramer. The N-terminal domain binds DNA with a considerably lower affinity than the full-length enzyme, and the interaction with DNA is significantly increased by the presence of ADPNP i.e. in the dimeric state. The presence of DNA does not appear to cause the enzyme to dimerise, nor does it induce a large conformational change in the enzyme. Taken together, these results help to clarify the role of the N-terminal domain in the catalytic cycle of human DNA topoisomerase Ilalpha. The hyperstimulation phenomenon, previously seen with the full-length enzyme, is now known to be a property of the N-terminal domain, and suggests a significant role for the T segment in the ATPase cycle of this enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available