Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence - PubMed (original) (raw)
Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence
A S Kimball et al. Nucleic Acids Res. 1995.
Free PMC article
Abstract
The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.
Similar articles
- Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination.
Chen JW, Evans B, Rosenfeldt H, Jayaram M. Chen JW, et al. Gene. 1992 Sep 21;119(1):37-48. doi: 10.1016/0378-1119(92)90064-v. Gene. 1992. PMID: 1398089 - DNA sequence determinant for FIp-induced DNA bending.
Luetke KH, Sadowski PD. Luetke KH, et al. Mol Microbiol. 1998 Jul;29(1):199-208. doi: 10.1046/j.1365-2958.1998.00922.x. Mol Microbiol. 1998. PMID: 9701814 - Analyses of the first chemical step in Flp site-specific recombination: Synapsis may not be a pre-requisite for strand cleavage.
Voziyanov Y, Lee J, Whang I, Lee J, Jayaram M. Voziyanov Y, et al. J Mol Biol. 1996 Mar 8;256(4):720-35. doi: 10.1006/jmbi.1996.0120. J Mol Biol. 1996. PMID: 8642593 - Application of FLP/FRT site-specific DNA recombination system in plants.
Luo H, Kausch AP. Luo H, et al. Genet Eng (N Y). 2002;24:1-16. doi: 10.1007/978-1-4615-0721-5_1. Genet Eng (N Y). 2002. PMID: 12416298 Review. No abstract available. - Recombination. Pieces of the site-specific recombination puzzle.
Oram M, Szczelkun MD, Halford SE. Oram M, et al. Curr Biol. 1995 Oct 1;5(10):1106-9. doi: 10.1016/s0960-9822(95)00222-3. Curr Biol. 1995. PMID: 8548278 Review.
Cited by
- Gene-targeting technologies for the study of neurological disorders.
Beglopoulos V, Shen J. Beglopoulos V, et al. Neuromolecular Med. 2004;6(1):13-30. doi: 10.1385/NMM:6:1:013. Neuromolecular Med. 2004. PMID: 15781974 Review. - Target DNA structure plays a critical role in Tn7 transposition.
Kuduvalli PN, Rao JE, Craig NL. Kuduvalli PN, et al. EMBO J. 2001 Feb 15;20(4):924-32. doi: 10.1093/emboj/20.4.924. EMBO J. 2001. PMID: 11179236 Free PMC article. - A histone octamer blocks branch migration of a Holliday junction.
Grigoriev M, Hsieh P. Grigoriev M, et al. Mol Cell Biol. 1997 Dec;17(12):7139-50. doi: 10.1128/MCB.17.12.7139. Mol Cell Biol. 1997. PMID: 9372946 Free PMC article. - Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity.
Lee J, Tonozuka T, Jayaram M. Lee J, et al. Genes Dev. 1997 Nov 15;11(22):3061-71. doi: 10.1101/gad.11.22.3061. Genes Dev. 1997. PMID: 9367987 Free PMC article.
References
- Q Rev Biophys. 1990 Aug;23(3):205-80 - PubMed
- Biochemistry. 1990 May 22;29(20):4747-51 - PubMed
- EMBO J. 1991 Sep;10(9):2589-94 - PubMed
- Mol Cell Biol. 1991 Sep;11(9):4497-508 - PubMed
- Cell. 1991 Nov 1;67(3):517-28 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources