Abstract
The RecA protein of Escherichia coli is essential for genetic recombination and has been extensively characterized (1–3). In vitro, purified RecA protein is able to promote recombination reactions of two types: (i) strand transfer between circular single-stranded DNA (ssDNA) and homologous linear duplex DNA and (ii) strand exchange between circular duplex DNA with a defined single-stranded gap and homologous linear duplex DNA (Fig. 1A) In this chapter, we describe the preparation of substrates for reaction (ii), which occurs between essentially duplex DNA molecules. The reaction has been used extensively in studies of the mechanism of RecA-mediated strand exchange (4,5) and may also be used to assay for activities capable of resolving Holliday junctions in DNA (6,7).
(A) Schematic drawing showing the substrates (gDNA and PsrI-linearized duplex DNA of 0X174) (left), intermediates (center), and the products (nicked circular duplex DNA and gapped linear duplex DNA) (right) of RecA-mediated strand exchange. The position of the 3′ [32P]-end-label is indicated (*).
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© 1994 Humana Press Inc.
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Miiller, B., West, S.C. (1994). An Assay for In Vitro Recombination Between Duplex DNA Molecules. In: Geoff Kneale, G. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 30. Humana Press. https://doi.org/10.1385/0-89603-256-6:413
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DOI: https://doi.org/10.1385/0-89603-256-6:413
Publisher Name: Humana Press
Print ISBN: 978-0-89603-256-9
Online ISBN: 978-1-59259-517-4
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