How do type II topoisomerases use ATP hydrolysis to simplify DNA topology beyond equilibrium? Investigating the relaxation reaction of nonsupercoiling type II topoisomerases.
DNA topoisomerases control the topology of DNA (e.g., the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e., more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower-than-equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analyzed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the supercoil-sensing C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topoisomerase VI (which is only distantly related to type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range approximately 2-9 kbp and is not altered by reducing the free energy available from ATP hydrolysis by varying the ADP:ATP ratio. A direct test of one model (DNA tracking; i.e., sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect, but that it is possible that other kinetic factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.
Stuchinskaya
Tanya
T
Department of Biological Chemistry, John Innes Centre, Colney, Norwich, UK.
Mitchenall
Lesley A
LA
Schoeffler
Allyn J
AJ
Corbett
Kevin D
KD
Berger
James M
JM
Bates
Andrew D
AD
Maxwell
Anthony
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D010957:Plasmids
D005656:Fungal Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II