Linked Life Data

10748189

Source:http://linkedlifedata.com/resource/pubmed/id/10748189

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
26
pubmed:dateCreated
2000-8-10
pubmed:abstractText
Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
30
pubmed:volume
275
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
19498-504
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:10748189-Adenosine Triphosphate, pubmed-meshheading:10748189-Binding Sites, pubmed-meshheading:10748189-Catalysis, pubmed-meshheading:10748189-DNA, Superhelical, pubmed-meshheading:10748189-DNA Topoisomerases, Type I, pubmed-meshheading:10748189-DNA Topoisomerases, Type II, pubmed-meshheading:10748189-Dose-Response Relationship, Drug, pubmed-meshheading:10748189-Escherichia coli, pubmed-meshheading:10748189-Mutagenesis, Site-Directed, pubmed-meshheading:10748189-Mutation, pubmed-meshheading:10748189-Oligonucleotides, pubmed-meshheading:10748189-Protein Conformation, pubmed-meshheading:10748189-Protein Structure, Tertiary, pubmed-meshheading:10748189-Recombinant Proteins, pubmed-meshheading:10748189-Silver Staining, pubmed-meshheading:10748189-Sodium Chloride, pubmed-meshheading:10748189-Sulfolobus acidocaldarius, pubmed-meshheading:10748189-Temperature, pubmed-meshheading:10748189-Tyrosine
pubmed:year
2000
pubmed:articleTitle
Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling.
pubmed:affiliation
Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Bât. 400, Université de Paris Sud, Centre d'Orsay, 91 405 Orsay Cedex, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't