Limits...
Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

Olorunniji FJ, McPherson AL, Pavlou HJ, McIlwraith MJ, Brazier JA, Cosstick R, Stark WM - Nucleic Acids Res. (2015)

Bottom Line: After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex.The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function.Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK.

Show MeSH
Nicked-site I substrates. (A) Left: a cartoon illustrating the generic structure of a nicked-site I. The symbols are as in Figure 1. Right: design of nicked-site I substrates. The site I sequence is shown boxed. The points at which resolvase breaks and rejoins the DNA strands are indicated by the staggered line. The positions of nicks in the bottom strand are indicated by arrows. (B) Products of resolvase-mediated cleavage. The substrates used here can be classified into four types as shown in the left-hand column. The products of cleavage of one strand are shown in the centre column, and products of double strand cleavage are in the right-hand column. (The n-site I products are shown again in the bottom panel of the right-hand column, for completeness.) Symbols are as in Figure 1; additionally, the central 2 bp of the sites is shown as short vertical lines, and attached resolvase subunits are shown as filled circles. 5′ phosphate groups at the positions of the nicks are indicated by small unfilled circles. The asterisks indicate the position of the 32P radioactive label at the left end of the site as in experiments shown in Figures 3–7.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499144&req=5

Figure 2: Nicked-site I substrates. (A) Left: a cartoon illustrating the generic structure of a nicked-site I. The symbols are as in Figure 1. Right: design of nicked-site I substrates. The site I sequence is shown boxed. The points at which resolvase breaks and rejoins the DNA strands are indicated by the staggered line. The positions of nicks in the bottom strand are indicated by arrows. (B) Products of resolvase-mediated cleavage. The substrates used here can be classified into four types as shown in the left-hand column. The products of cleavage of one strand are shown in the centre column, and products of double strand cleavage are in the right-hand column. (The n-site I products are shown again in the bottom panel of the right-hand column, for completeness.) Symbols are as in Figure 1; additionally, the central 2 bp of the sites is shown as short vertical lines, and attached resolvase subunits are shown as filled circles. 5′ phosphate groups at the positions of the nicks are indicated by small unfilled circles. The asterisks indicate the position of the 32P radioactive label at the left end of the site as in experiments shown in Figures 3–7.

Mentions: Binding/synapsis assays were based on previously described methods (7). Briefly, substrates containing site I (the Tn3 crossover site) were assembled by annealing equimolar amounts of 50 nt (or 70 nt) top and bottom strand oligonucleotides, one of which was first 32P-labelled at the 5′ end. The nicked-site substrates used in single-strand cleavage assays were assembled by annealing a 50 nt or a 70 nt site I top strand oligonucleotide with equimolar amounts of two shorter bottom strand oligonucleotides, such that a single-strand break (nick) is present near or at the scissile position on the bottom strand, and the assembled double-stranded substrate has blunt ends. The appropriate oligonucleotide was 32P-labelled at its 5′ end with T4 polynucleotide kinase. The top strand of the 70 bp site is 5′-ggcaagcttgcgtgactcaacTGTCTGATAATTTATAAATTATCAGACAtagtgggatggtctgcagcgg-3′; the central AT dinucleotide of site I is in bold, and the nucleotides flanking site I are in lower case. The 50 bp site is 10 bp shorter at each end (lacking the nucleotides in italics). The positions of bottom strand nicks are described in the main text. The 5′ end at the nick is phosphorylated unless stated otherwise (see Figure 2A).


Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

Olorunniji FJ, McPherson AL, Pavlou HJ, McIlwraith MJ, Brazier JA, Cosstick R, Stark WM - Nucleic Acids Res. (2015)

Nicked-site I substrates. (A) Left: a cartoon illustrating the generic structure of a nicked-site I. The symbols are as in Figure 1. Right: design of nicked-site I substrates. The site I sequence is shown boxed. The points at which resolvase breaks and rejoins the DNA strands are indicated by the staggered line. The positions of nicks in the bottom strand are indicated by arrows. (B) Products of resolvase-mediated cleavage. The substrates used here can be classified into four types as shown in the left-hand column. The products of cleavage of one strand are shown in the centre column, and products of double strand cleavage are in the right-hand column. (The n-site I products are shown again in the bottom panel of the right-hand column, for completeness.) Symbols are as in Figure 1; additionally, the central 2 bp of the sites is shown as short vertical lines, and attached resolvase subunits are shown as filled circles. 5′ phosphate groups at the positions of the nicks are indicated by small unfilled circles. The asterisks indicate the position of the 32P radioactive label at the left end of the site as in experiments shown in Figures 3–7.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499144&req=5

Figure 2: Nicked-site I substrates. (A) Left: a cartoon illustrating the generic structure of a nicked-site I. The symbols are as in Figure 1. Right: design of nicked-site I substrates. The site I sequence is shown boxed. The points at which resolvase breaks and rejoins the DNA strands are indicated by the staggered line. The positions of nicks in the bottom strand are indicated by arrows. (B) Products of resolvase-mediated cleavage. The substrates used here can be classified into four types as shown in the left-hand column. The products of cleavage of one strand are shown in the centre column, and products of double strand cleavage are in the right-hand column. (The n-site I products are shown again in the bottom panel of the right-hand column, for completeness.) Symbols are as in Figure 1; additionally, the central 2 bp of the sites is shown as short vertical lines, and attached resolvase subunits are shown as filled circles. 5′ phosphate groups at the positions of the nicks are indicated by small unfilled circles. The asterisks indicate the position of the 32P radioactive label at the left end of the site as in experiments shown in Figures 3–7.
Mentions: Binding/synapsis assays were based on previously described methods (7). Briefly, substrates containing site I (the Tn3 crossover site) were assembled by annealing equimolar amounts of 50 nt (or 70 nt) top and bottom strand oligonucleotides, one of which was first 32P-labelled at the 5′ end. The nicked-site substrates used in single-strand cleavage assays were assembled by annealing a 50 nt or a 70 nt site I top strand oligonucleotide with equimolar amounts of two shorter bottom strand oligonucleotides, such that a single-strand break (nick) is present near or at the scissile position on the bottom strand, and the assembled double-stranded substrate has blunt ends. The appropriate oligonucleotide was 32P-labelled at its 5′ end with T4 polynucleotide kinase. The top strand of the 70 bp site is 5′-ggcaagcttgcgtgactcaacTGTCTGATAATTTATAAATTATCAGACAtagtgggatggtctgcagcgg-3′; the central AT dinucleotide of site I is in bold, and the nucleotides flanking site I are in lower case. The 50 bp site is 10 bp shorter at each end (lacking the nucleotides in italics). The positions of bottom strand nicks are described in the main text. The 5′ end at the nick is phosphorylated unless stated otherwise (see Figure 2A).

Bottom Line: After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex.The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function.Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK.

Show MeSH