Alexander L. Nussbaum

Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate.

In order to study the interactions of the EcoRI restriction endonuclease and modification methylase with DNA, we have synthesized the self-complementary octanucleotide, pT-G-A-A-T-T-C-A, which contains the nucleotide sequence of the EcoRI substrate. This octamer can act as a substrate for both the endonuclease and methylase; the enzymatic alteration of this molecule is the same as that of DNA, with cleavage and methylation both occurring at the same positions on either substrate. The optimum temperature for the reaction of the octanucleotide with the endonuclease is 15°C and that with the methylase is 12·5°C. The optimum temperature for the reactions of both of the enzymes with DNA is 37°C. The low temperatures for reactions with the octanucleotide reflect the conditions under which this molecule can serve as a substrate for the enzymes. The Km values and turnover numbers for the EcoRI endonuclease using both the synthetic octanucleotide and simian virus 40 (SV40) DNA as substrates were determined. These constants are, respectively, 7 × 10−6 m and 4 min−1 for the endonuclease-octamer reaction and 3 × 10−8 m and 3 min−1 with SV40 DNA. Although the Km does not measure affinity directly, the similarity of turnover numbers on both substrates allows us to conclude that the affinity of the enzyme for SV40 DNA is approximately 200 times greater than for the octanucleotide. The synthetic octanucleotide is self-complementary and can form a duplex structure. Computer analysis of initial reaction-rate data shows that in order to act as a substrate the octamer must be a dimer. This observation, together with the correspondence of the temperature optima for both enzymatic reactions to the tm value for the double-stranded form of the octanucleotide (17 to 19°C), lead us to conclude that the substrate for both EcoRI enzymes is a double-helical segment of DNA containing the central hexamer of the octanucleotide. It is unlikely that any cruciform rearrangement is required for enzyme activity.

Small-scale preparation of 5′-nucleotides and analogs by carrot phosphotransferase

Abstract Carrot phosphotransferase has been used to prepare a variety of 5′-nucleotides and analogs for E. coli DNA polymerase binding site studies. The ability of the enzyme to ignore a wide variety of structural variation and stereochemical detail, while preserving its specificity in substrate phosphorylation, makes it a useful tool in such preparations. The activity has been further characterized by kinetic studies.

Chemical synthesis of a tetradecamer in the deoxyribonucleic acid series

Abstract The chemical synthesis of a tetradecadeoxyribonucleotide, d-EtSp(A-T-G-G-A-A-A-C-T-G-C-G-G-C), is described. This oligomer, designated Fragment 4δ, constitutes the 5′-terminus of the plus strand of a projected duplex coding for S-Peptide 2–14 derived from Ribonuclease A. The Fragment was constructed by block condensation via a phosphorothioate anchor. Complications due to inadvertent phosphotriester condensations are discussed. Arguments justifying the sequence selection are presented.

Chain length characterization of oligodeoxyribonucleotides by analytical ultracentrifugation.

Abstract Analytical ultracentrifugation was used to determine chain length of oligomers in the deoxyribonucleotide series. Uncertainties with respect to partial specific volume of the solute and questions of charge compel a semiempirical approach: variations of these parameters introduce fluctuations well within the experimental error of the method. Measurements of the sedimentation equilibria for three homooligomer series—d(pT)n, d(pA)n and d(pC)n—as well as a number of oligomers of varying base composition generated straight-line plots passing through the origin.

Sequential cleavage of dinucleotides from DNA by SP3 DNase: III. Substrate specificity and initiation of the hydrolysis from the 5′-Terminus of polynucleotides☆☆☆

Abstract SP3 DNase, an enzyme potentially useful for DNA sequence studies, has the unusual property of cleaving dinucleotides from single-stranded DNA. The present paper describes the action of this enzyme on synthetic oligo- and polydeoxyribonucleotides. These studies elucidate the specificity and mode of action of this enzyme. Polydeoxythymidylate and polydeoxyadenylate are hydrolyzed completely to dinucleotides by SP3 DNase. Although polydeoxycytidylate is hydrolyzed to yield mostly d(pC) 2 , small amounts of d(pC) 4 are also produced. 5-Methylpolyde-oxycytidylic acid is also hydrolyzed by SP3 DNase. The enzyme does not use polyribonucleotides as substrates. The enzyme does not hydrolyze dimers or trimers. For example, d(pTpT), d(pApA), d(pCpC), d(pTpTpT), d(pApApA) or d(pCpCpC) are not hydrolyzed. Tetramers of the general structure d(pXpXpXpX) are hydrolyzed to dimers. The tetramer d(pT) 4 is completely converted to d(pT) 2 . However, d(pA) 4 and d(pC) 4 are not hydrolyzed completely even in the presence of larger amounts of enzyme. The pentamer, d(pT) 5 , is hydrolyzed by the enzyme to yield the dimer, d(pT) 2 , plus the trimer, d(pT) 3 . The hexamer and octamer of deoxythymidylate are converted completely to the dimer. Both Mg 2+ and Mn 2+ are required for the action of the enzyme on d(pT) 4 but when the pentamer and longer polymers of deoxythymidylate are substrates, the presence of Mn 2+ is no longer necessary. Although d(pT) 4 is hydrolyzed, d(TpTpTpT) is not. The tetramer terminated by a 3′-phosphate and lacking a 5′-phosphate inhibits the enzyme. This indicates that a tetramer must have a terminal 5′-phosphate to serve as substrate. A terminal 5′-phosphate is not essential, however, for pentamer hydrolysis; d(TpTpTpTpT) is hydrolyzed to d(TpT) plus d(pTpTpT). Contrary to preliminary kinetic data, SP3 DNase initiates its attack from the 5′-terminus of a polydeoxynucleotide strand. Unequivocal experiments demonstrate that 8P3 DNase hydrolyzes d(pTpTpTpTpA) to d(pTpT) and d(pTpTpA). Confirmation of the attack from the 5′-terminus was shown by the hydrolysis of d(pApTpTpTpT) to d(pApT) and d(pTpTpT). The potential use of the enzyme for DNA nucleotide sequence studies is discussed.

Gel filtration of acylated oligonucleotides

The behavior of a number of acylated deoxyribooligonucleotides in gel permeation chromatography has been studied and dependence on factors other than molecular size has been noted. Retardation has been observed to increase as follows: pT < d-pABz < d-pCAn < d-pGiBu, and this order is reflected in the elution parameters of derived oligomers. Certain nucleotide derivatives—notably d-pCAn and its relatives—were eluted in two peaks.