THE ROLE OF SINGLE-STRANDED-DNA IN FLP-MEDIATED STRAND EXCHANGED

Citation
Xd. Zhu et Pd. Sadowski, THE ROLE OF SINGLE-STRANDED-DNA IN FLP-MEDIATED STRAND EXCHANGED, The Journal of biological chemistry, 273(9), 1998, pp. 4921-4927
Citations number
46
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
273
Issue
9
Year of publication
1998
Pages
4921 - 4927
Database
ISI
SICI code
0021-9258(1998)273:9<4921:TROSIF>2.0.ZU;2-T
Abstract
The Flp recognition target site contains two inverted 13-base pair (bp ) Flp binding sequences that surround an 8-bp core region. Flp recombi nase has been shown to carry out strand ligation independently of its ability to execute strand cleavage. Using a synthetic activated DNA su bstrate bearing a S'-phosphotyrosine group, we have developed an assay to measure strand exchange by Flp proteins. We have shown that wild-t ype Flp protein was able to catalyze strand exchange using DNA substra tes containing 8-bp duplex core sequences. Mutant Flp proteins that ar e defective in either DNA bending or DNA cleavage were also impaired i n their abilities to carry out strand exchange. The inability of these mutant proteins to execute strand exchange could be overcome by provi ding a DNA substrate containing a single-stranded core sequence. This single-stranded core sequence could be as small as 3 nucleotides. Full activity of mutant Flp proteins in strand exchange was observed when both partner DNAs contained an 8-nucleotide single-stranded core regio n. Using suicide substrates, we showed that single-stranded DNA is als o important for strand exchange reactions where Pip-mediated strand cl eavage is required. These results suggest that the ability of Flp to i nduce DNA bending and strand cleavage may be crucial for strand, excha nge. We propose that both DNA bending and strand cleavage may be requi red to separate the strands of the core region and that single-strande d DNA in the core region might be an intermediate in Pip-mediated DNA recombination.