Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites. A method is described whereby any Eco RI site can be targeted for specific cleavage. The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex. This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation. When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes. The method was tested on lambda phage, Escherichia coli, and human DNA. Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained.

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The conformations and binding site environments of Mg2+TTP and Mg2+dATP bound to Escherichia coli DNA polymerase I and its large (Klenow) fragment have been investigated by proton NMR. The effect of the large fragment of Pol I on the NMR line widths of the protons of Mg2+TTP detected one binding site for this substrate with a dissociation constant of 300 +/- 100 microM and established simple competitive binding of deoxynucleoside triphosphates at this site in accord with previous equilibrium dialysis experiments with whole Pol I [Englund, P. T., Huberman, J.A., Jovin, T.M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038]. Primary negative nuclear Overhauser effects were used to calculate interproton distances on enzyme-bound Mg2+dATP and Mg2+TTP. These distances established that each substrate was bound with an anti-glycosidic torsional angle (chi) of 50 +/- 10 degrees for Mg2+dATP and 40 +/- 10 degrees for Mg2+TTP. The sugar pucker of both substrates was predominantly O1'-endo, with a C5'-C4'-C3'-O3' exocyclic torsional angle (delta) of 95 +/- 10 degrees for Mg2+dATP and 100 +/- 10 degrees for Mg2+TTP. The consistency of these conformations with those previously proposed, on the basis of distances from Mn2+ at the active site [Sloan, D. L., Loeb, L. A., Mildvan, A.S., & Feldman, R.J. (1975) J. Biol. Chem. 250, 8913], indicates a unique conformation for each bound nucleotide. The chi and delta values of the bound substrates are appropriate for nucleotide units of B DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)