EFFECT OF A 3'-]5'-EXONUCLEASE WITH A PROOFREADING FUNCTION ON THE FIDELITY OF ERROR-PRONE DNA-POLYMERASE-ALPHA FROM REGENERATING LIVER OF AGED RATS

A nuclease that releases noncomplementary nucleotides from the 3'-end of DNA was isolated and highly purified from rat liver extract. The d( T-9-C) priming activities for DNA synthesis in vitro by DNA polymerase s alpha and beta were recovered by the addition of this enzyme, which itself does not contain a DNA polymerase activity. This nuclease hydro lysed nucleotides from the 3'-end, but did not remove [P-32]-labeled n ucleotides from the 5'-terminus of specifically labeled DNA. Also, the reaction products released from the 3'-end of DNA were all mononucleo tides. These results indicate that the exonuclease is a 3'-->5' exonuc lease with properties the same as those of DNase VII from human placen ta. Rat DNase VII requires 4 mM MgCl2 or 0.125 mM MnCl2 for maximum ac tivity, and shows a pH optimum of 7.5. These optimal conditions are si milar to those of DNA polymerases, and indicate that both rat DNase VI I and DNA polymerases are able to act under same conditions. Non-compl ementary nucleotide incorporation by DNA polymerase a from aged rat ha s been observed during in vitro DNA synthesis on poly dA-dT(10). The a mount of this mis-incorporation is decreased by the coexistence of the 3'-->5' exonuclease, but not all errors are edited out. Thus, this ra t DNase VII is suggested to play an important role in proofreading dur ing DNA synthesis. (C) 1998 Elsevier Science Ireland Ltd.