DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang

Abstract

The specificity of vaccinia topoisomerase for transesterification to DNA at the sequence 5'-CCCTT and its versatility in strand transfer have illuminated the recombinogenic properties of type IB topoisomerases and spawned topoisomerase-based strategies for DNA cloning. Here we characterize a pathway of topoisomerase-mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide rapidly and efficiently joins the CCCTT strand to a duplex DNA containing a 3' A overhang. The joining reaction occurs with high efficiency, albeit slowly, to duplex DNAs containing 3' G, T or C overhangs. Strand transfer can be restricted to the correctly paired 3' A overhang by including 0.5 M NaCl in the ligation reaction mixture. The effects of base mismatches and increased ionic strength on the rates of 3' overhang ligation provide a quantitative picture of the relative contributions of +1 T:A base pairing and electrostatic interactions downstream of the scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The results clarify the biochemistry underlying topoisomerase-cloning of PCR products with non-templated 3' overhangs.

Figure 1

Cleavage substrate and 3′ N strand transfer acceptors. The CCCTT-containing substrate is shown with the site of cleavage indicated by the arrow and the 5′ ³²P-label on the 60mer strand denoted by the asterisk. Transesterification by topoisomerase to the scissile phosphate results in formation of the indicated covalent intermediate and dissociation of the downstream 3′-tailed duplex leaving group. A series of acceptor DNAs containing 3′ N overhangs is shown. Attack of either 5′ OH of the acceptor DNA on the covalent intermediate results in strand transfer to yield a mixture of two recombinant molecules shown. The ³²P-labeled strand of the recombinant product is 54 nt long.

Figure 2

Topoisomerase-catalyzed strand transfer at a 3′ T:A overhang. The DNA cleavage and strand transfer reactions and product analyses were performed as described in Materials and Methods. An autoradiograph of the polyacrylamide gel is shown. The reaction times are specified above the lanes. A control reaction mixture containing the substrate, but lacking topoisomerase, was analyzed in the leftmost lane. The positions of the radiolabeled input 60mer scissile strand, the 54mer recombinant product, and the covalent DNA–peptide adduct are indicated on the right.

Figure 3

Strand transfer with mispaired 3′ overhangs. The DNA cleavage and strand transfer reactions and product analyses were performed as described in Materials and Methods. The strand transfer reaction mixtures were incubated at 37°C for 1 h (acceptor A), 2 h (acceptors G and T) or 3 h (acceptor C). An autoradiograph of the polyacrylamide gel is shown. A mixture containing the substrate, but lacking topoisomerase, was analyzed in the leftmost lane. A cleavage reaction containing topoisomerase was analyzed in lane –. The positions of the radiolabeled input 60mer scissile strand, the 54mer strand transfer product and the covalent DNA–peptide adduct are indicated on the right.

Figure 4

Kinetics of 3′ overhang ligation. The cleavage and strand transfer reactions were performed as described in Materials and Methods. Strand transfer was initiated by addition of a 50-fold molar excess of the indicated 3′ N acceptor DNA (A) or simultaneous addition of a 50-fold molar excess of the indicated 3′ N acceptor DNA plus NaCl to attain a final salt concentration of 0.5 M (B). Aliquots were withdrawn at the times specified. The proteinase K-digested reaction products were analyzed by polyacrylamide gel electrophoresis. The extent of formation of the 54mer recombinant strand (as a percent of the total radioactivity) is plotted as a function of time.

Figure 5

Salt inhibition of 3′ mismatch ligation. Strand transfer was initiated by addition of a 50-fold molar excess of the indicated 3′ N acceptor DNA plus NaCl to attain final salt concentrations as specified. The reactions were quenched after 1 h (acceptor A) or 2 h (acceptor G) at 37°C. The extent of formation of the 54mer recombinant strand (as a percent of the total radioactivity) is plotted as a function of NaCl concentration.

Figure 6

Transfer of a covalently bound 3′ T overhang to a blunt-end acceptor DNA. The cleavage and strand transfer reactions were performed as described in Figure 2. Strand transfer was initiated by addition of a 50-fold molar excess of the indicated blunt-end 24 bp acceptor DNA or simultaneous addition of a 50-fold molar excess of blunt-end acceptor DNA plus NaCl to attain a final salt concentration of 0.5 M. Aliquots were withdrawn at the times specified. The extent of formation of the 54mer recombinant strand (as a percent of the total radioactivity) is plotted as a function of time.