Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated exonuclease activities.

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.

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