Abstract
The combined action of exonuclease I and recA protein leads to a kind of reverse DNA strand exchange in which joint molecules formed on the "wrong" or distal end of a linear duplex in the presence of ATP are stabilized by exonuclease I degradation of the displaced (+) strand. Continued pairing and degradation of the displaced strand leads to strand exchange that appears to progress with a polarity opposite that of the normal recA protein promoted reaction (i.e. 3'-5' with respect to the (+) strand). However, in contrast to the normal 5'-3' strand exchange, the displaced strand is completely degraded in the process. When the linear duplex DNA substrate has a heterologous region at the 5' (proximal) end, the major product (described in a previous study (Bedale, W. A., Inman, R. B., and Cox, M. M. (1991) J. Biol. Chem. 266, 6499-6510)) is a circular duplex DNA molecule with a double-stranded tail whose length corresponds closely to the heterologous segment of the substrate. The origin of this product is here shown to be the result of the exonuclease activity of exonuclease I (either added exogenously or present as a trace contaminant of recA protein or SSB protein preparations), as opposed to endonucleolytic or mechanical breakage. The levels of exonuclease I required to generate these products are sufficiently low that they are undetected by assays for exonuclease contamination in recA protein preparations. These results demonstrate that the interplay of recA protein with other enzymes can have a profound effect on both the mechanism and outcome of recA protein-promoted DNA strand exchange. They also demonstrate that the (+) strand of the duplex DNA substrate is at least transiently displaced in recA protein-mediated pairing even when joint molecules are limited to the distal end.
Highlights
From the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
In which joint molecules formed on the “wrong” or protein of E. coli is included in the invitro reactions and distal end of a linear duplex in the presence of ATP probably facilitates formation of a uniform and stable filaare stabilized by exonuclease I degradation of the dis- ment of recA protein on the ssDNA’ (Cox et al, 1987; Koplaced (+) strand. continued pairing and degradation of the displaced strand leads to strand exchange that appearsto progress with a polaroitpyposite that of the normal recA protein promoted reaction (i.e. 3‘-5’with respect to the (+) strand)
(+) strand of the duplex DNA substrate is at least transiently displaced in recA protein-mediated pairing even when joint moleculesare limited to the distaelnd
Summary
(Received for publication, November 5, 199a2n,d in revised form, March 23, 1993). From the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706. While this paper was in preparation, Konforti and Davis (1992) reported that the addition of exonuclease I to recA protein-mediated DNA strand exchange reactions facilitates the formation of joint molecules containing short regions of homology atthe 3' end of the (+) strandand presented evidence suggesting that the 3' end of the (+) strand of the duplex had been displaced to some degree. A recA protein preparation was identified that lacked the ability to generate a-like molecules in reactions where the duplex DNA substrate had 5' heterologous sequences, but was still fully active in promoting strand exmM potassium glutamate, 1 mM dithiothreitol, 5% glycerol, and an change and jointmolecule formation between completely ho-. The mechcopy were performed as described previously (Bedale et al, 1991). anism bywhich the contaminating nuclease facilitates formation of a-like molecules wasaddressed by electron micros-
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