Linked Life Data

8652567

Source:http://linkedlifedata.com/resource/pubmed/id/8652567

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
1996-7-30
pubmed:abstractText
The monomeric Escherichia coli Rep protein undergoes a DNA-induced dimerization upon binding either single-stranded (ss) or duplex DNA with the dimer being the active form of the Rep helicase. Using stopped-flow fluorescence, we have determined a minimal kinetic mechanism for this reaction in which Rep monomer (P) binds to ss oligodeoxynucleotides (dN(pN)15) (S) by a two-step mechanism to form PS*, which can then dimerize with P to form P2S as indicated: [reaction in text]. This minimal mechanism is supported by four independent studies in which the kinetics were monitored by changes in fluorescence intensity of three different probes: the intrinsic Rep tryptophan fluorescence, the fluorescence of d(T5(2-AP)T4(2-AP)T5), containing the fluorescent base, 2-aminopurine (2-AP), and dT(pT)15 labeled at its 3'-end with fluorescein (3'-F-dT(pT)15). Simultaneous (global) analysis of the time courses of d(T5(2-AP)T4(2-AP)T5) (100 nM) binding to a range of Rep monomer concentrations (25-400 nM) yields the following rate constants: k1 = (3.3 +/- 0.5) x 10(7) M-1 s-1; k-1 = 1.4 +/- 0.4 s-1; k2 = 2.7 +/- 0.9 s-1; k-2 = 0.21 +/- 0.06 s-1; k3 = (4.5 +/- 0.3) x 10(5) M-1 s-1; k-3 = 0.0027 +/- 0.0008 s-1 [20 mM Tris-HCl, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4.0 degrees C]. This mechanism provides direct evidence that Rep monomers can bind ss DNA and that ss DNA binding induces a conformational change in the Rep monomer that is probably required for Rep dimerization. This conformational change is likely to be large and global since it is detected by all three fluorescence probes. The apparent bimolecular rate constant for Rep monomer binding to 3'-F-dT(pT)15 [k1(app) = (6.0 +/- 0.7) x 10(7) M-1 s-1] is slightly larger than measured with d(T5(2-AP)T4(2-AP)T5) binding. The apparent rate constant for dissociation of d(T5(2-AP)T4(2-AP)T5) (S) from the half-ligated Rep dimer, P2S, increases with increasing concentration of a nonfluorescent competitor ss DNA (d(T5-AT4AT5)) (C), indicating transient formation of a doubly ligated P2SC intermediate. However, the apparent bimolecular rate constant for binding of C to P2S is extremely slow (> or = 250 M-1 s-1), suggesting the occurrence of a multistep process before dissociation of ss DNA. In the absence of competitor DNA, dissociation of ss DNA from P2S occurs only after slow dissociation of the Rep dimer to form PS* + P. The implications of these results for Rep-catalyzed DNA unwinding are discussed.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2268-82
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Kinetic mechanism of DNA binding and DNA-induced dimerization of the Escherichia coli Rep helicase.
pubmed:affiliation
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't