Abstract
Purified DNA polymerase III has two distinct exonuclease activities: one initiates hydrolsis at the 3 termini, and the other at the 5 termini of single-stranded DNA. Both exonucleases have the same relative mobility on polyacrylamide gels as the polymerase activity. Molecular identity of the three activities is further indicated by their comparative rates of thermal inactivation and their sensitivity to ionic strength. The 3-5 exonuclease activity hydrolyzes only single-standed DNA. The rate of hydrolysis is twice the optimal rate of polymerization. The products are 5-mononucleotides, but the 3-5 activity is unable to cleave free dinucleotides or the 5-terminal dinucleotide of a polydeoxynucleotide chain. The 3-5 activity will not degrade 3-phosphoryl-terminated oligonucleotides such as d(pTpTpTp). The 5-3 activity catalyzes the hydrolysis of single-stranded DNA at 1/15 the rate of the 3-5 exonuclease. The 5-3 exonuclease requires the presence of a 5 single-stranded terminus in order to initiate hydrolysis, but will thereafter proceed into a double-stranded region. Although the limit products found during hydrolysis of substrates designed to assay specifically the 5-3 activity are predominantly mono- and dinucleotides, these products probably arise from the subsequent hydrolysis of oligonucleotides by the 3-5 hydrolytic activity. This interpretation is supported by (a) the relatively greater activity of the 3-5 exonuclease, (b) the inability of the enzyme to degrade d(pTpTpTp), and (c) the release of the 5 terminus of a single-stranded DNA molecule as an oligonucleotide. The 5-3 exonuclease attacks ultraviolet-irradiated duplex DNA which has first been incised by the Micrococcus luteus endonuclease specific for thymine dimers in DNA.
Highlights
After the incubation with pancreatic 1)Nase the reaction mixture was boiled for 5 min
Other single-stranded linear molecules serve as substrates (Table I). l’olymerase 111 hydrolyzes exenucleasc II I-treated I)NA, which has 5’ but not 3’ single-stranded termini, at >f5 the rate of the same DNA which has been heatdenatured
I)NA prepared by incubation uith micrococcal nuclease
Summary
Preparation of [3H,5’-32P]d(pTpZ’),[3H,5’-32P]d(pTpTpT),[3H 5’-32P]d(p2’pTpTpT), and [3H,6’-32t)]d(pTpTpTpTpT)- [3H]-pol;(dT) was digested to greater than 957; acid solubility with pancreatic DNase as previously described [17]. After the incubation with pancreatic 1)Nase the reaction mixture was boiled for 5 min. The 5’.phosphoryl groups of the products were removed by incubating 200 nmol of oligonucleotides in a reaction mixture COII-. Taining 0.1 M Tris-IICl buffer (pH 8.0), 10 mM MgClz, and 0.25 unit of bacterial alkaline phosphatase. 16.7 mM in Tris-HCI buffer (pH 7.6), 10 mM in MgClz, 16.7 rnM in. Ten units of polynucleotide kinase were added, and the reaction mixture was incubated at 37” for 30 min. 1 N HCl was added to make the solution 0.2 N and the reaction mixture was placed in boiling water for 30 min to hydrolyze the ATP. The reaction was titrated to pH 7 with 1 M Tris base and diluted 50.fold with 7 M urea-5 mM Tris-HCl buffer (pH 7.6)
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