Linked Life Data

14757053

Source:http://linkedlifedata.com/resource/pubmed/id/14757053

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2004-2-3
pubmed:abstractText
The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3'-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the "closed" conformation when bound to a 3'-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3'-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3'-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0022-2836
pubmed:author
pubmed:issnType
Print
pubmed:day
13
pubmed:volume
336
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
395-408
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes.
pubmed:affiliation
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.