Abstract
During purification of a strand exchange activity from Schizosaccharomyces pombe using the three-strand reaction of double-stranded linear and circular single-stranded DNA, we identified p190/210 as an activity that stimulated the strand exchange activity of p140exo2 by about 10-fold. The accompanying report (Käslin, E., and Heyer, W.-D. (1994) J. Biol. Chem. 269, 0000-0000) described the purification and characterization of p140exo2, likely to be the S. pombe homolog of the Saccharomyces cerevisiae strand exchange protein p175SEP1. Here, we report the purification of p190/210 from S. pombe cells and its identification as fatty acid synthase (FAS). S. pombe FAS (p190/210) binds to single-stranded and double-stranded DNA, leading to condensation of DNA into large aggregates. In addition, it is capable of renaturing complementary single-stranded DNA. Besides stimulating the strand exchange activity of p140exo2, FAS (p190/210) itself exhibits strand exchange activity provided the double-stranded substrate has single-stranded tails. We propose a probable mechanism for the action of FAS (p190/210) during DNA strand exchange in vitro. Since FAS (p190/210) is highly unlikely to have a role in homologous recombination in vivo, we discuss the implications of our data on the interpretation of other homologous pairing and strand exchange proteins purified from eukaryotes using this or similar assays.
Highlights
During purificationof a strand exchange activfritoym lar to the Escherichia coli RecE a n d hRed pathways, wherethe
For someof the strand exchange activities, factors have been strandedDNA, we identifiedp190/210 as an activity thatpurified which stimulate in vitro strand exchange activity
We propose a probable mechanism for the actionYaorfrowia lipolytica FASl genes [12] and between thSe. cereui
Summary
Associated exonuclease activity comprising ~ 1 7 5 ~ ~fro' 'm Sac- Assay for Strand Exchange, Assayfor DNA Renaturation, and Eleccharomyces cerevisiae These systems mightbe simi- [2]. The reconstitution assay in which 5 pl of every fraction was SDS-polyacrylamide gel electrophoresis according Rtoef. 18using 0.75- assayed (instead of only 2 pl as in Fig. 1B)showed a weaker mm-thick gels in a Mighty small apparatus (Hoefer)
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