title: Mechanism of interaction between single-stranded DNA binding protein and DNA.
abstract: A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts with single-stranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coli and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively, as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where approximately 70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB x ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA gave no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to determine the dissociation constant for labeled SSB(2) x dT(70) to be 1.1 microM at a high ionic strength (200 mM NaCl). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced.
authors.affiliation: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Simone
authors.initials: S
authors.lastname: Kunzelmann
authors.forename: Caroline
authors.initials: C
authors.lastname: Morris
authors.forename: Alap P
authors.initials: AP
authors.lastname: Chavda
authors.forename: John F
authors.initials: JF
authors.lastname: Eccleston
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
mesh_terms: D002232:Carbocyanines / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D002232:Carbocyanines; D003374:Coumarins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C089187:cyanine dye 3; C088959:7-diethylaminocoumarin-3-carboxylic acid; D004265:DNA Helicases