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.