A reverse DNA strand exchange mediated by recA protein and exonuclease I. The generation of apparent DNA strand breaks by recA protein is explained.
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.
Bedale
W A
WA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Inman
R B
RB
Cox
M M
MM
D017104:Bacteriophage M13 / Q000235:genetics; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008854:Microscopy, Electron; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic
D004247:DNA; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I