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Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment.

Abstract：

:Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I. Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors to the donor fluorescein fluorophore used to modify cysteine 751 in the double mutant KF (S751C, C907S). Design of the template-primer allowed the probe's position within the DNA-protein complex to be varied by stepwise extension of the primer strand upon addition of the appropriate deoxynucleoside triphosphates (dNTP). The TMR acceptor probe occupied seven different positions in the template-primers, five in the single stranded region and two in the double stranded region. The efficiency of energy transfer was determined at each position by calculating the integrated area of the fluorescein emission peak in the presence and absence of acceptor. Results indicate that the FRET efficiency varied in a sinusoidal fashion with a periodicity of approximately 10 base pairs and that the data could be fitted to an equation derived from a simple model formulated on the basis of helical structure. The data support the conclusion that the single stranded template portion of a DNA template-primer adopts a helical conformation when bound to the KF. The results of this study further support FRET as a useful method for the determination of structure and conformation in protein-DNA complexes.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Furey WS,Joyce CM,Osborne MA,Klenerman D,Peliska JA,Balasubramanian S

doi

10.1021/bi9719758

subject

Has Abstract

pub_date

1998-03-03 00:00:00

pages

2979-90

issue

9

eissn

0006-2960

issn

1520-4995

pii

bi9719758

journal_volume

37

pub_type

杂志文章

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