Proof-reading 3'-->5' exonucleases isolated from rat liver nuclei.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-5) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A-T)] template, respectively, 10-fold and 2-fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polymerase activities being similar. Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.

[3'-->5'-exonucleases of the rat liver and the correction of replication errors]. / 3'-->5'-ékzonukleazy pecheny krysy i korrektsiia oshibok replikatsii.

Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes.

[Homogeneous 3'----5'-exonucleases and their multienzyme complexes from the rat liver]. / Gomogennye 3'----5'-ékzonukleazy i ikh mul'tifermentnye kompleksy iz pecheni krysy.

Isolation and general properties of 3'-5' exonucleases I and II (EC 3.1.4.26), which are specific to single-stranded DNA, are described. Such enzymes, being components of replication complexes, could correct replication errors. Homogeneous exonucleases I and II consist of a single subunit with molecular mass of 50 and 40 kDa, respectively. These enzymes are located preferentially in the nuclear membrane and chromatin. They form complexes with nuclear DNA polymerases and some other proteins and are not observed practically in a free state. Molecular masses of the complexes amount from 70 to 1.500 kDa. The complexes dissociate as a result of solution hydrophobization and can be reconstituted after the decrease of hydrophobization. The heavy membrane complex form of 3'----5' exonuclease I manifests enzymatic activities of DNA polymerase alpha (EC 2.7.7.7), non-specific nucleoside triphosphatase (EC 3.1.3.2), nucleotidase (EC 3.1.3.31) and faint activity of endonuclease (EC 3.1.4.5). Complexes under study do not display activity of thymidine kinase (EC 2.7.1.21), marker protein of replitase, neither in G0 nor in S-period.

[Complexes of nuclear DNA-polymerases with 3'----5'-exonucleases from the rat liver]. / Kompleksy iadernykh DNK-polimeraz c 3'----5'-ékzonukleazami iz pecheni krysy.

"Editing" 3'----5' exonuclease activity of DNA polymerases corrects replication errors. This activity associated with procaryotic DNA polymerases is not intrinsic to purified mammalian DNA polymerases. By means of extraction and subsequent gel filtration, several subspecies of complexes of 3'----5' exonuclease (E.C. 3.1.4.26) with DNA polymerases alpha, beta (E.C. 2.7.7.7) and some other proteins were isolated from chromatin, nucleoplasm, nuclear membrane, and cytosol. Complexes containing 3'----5' exonuclease manifest from 40 to 70% of total DNA polymerase activity revealed in different compartments of a hepatocyte. Molecular masses of the complexes amount from 250 to 1500 kDa They dissociate as a result of solution hydrophobization. DNA polymerase alpha activity enhances 5--8 folds during cell transition from G0 to S-period. The value of the ratio of 3'----5' exonuclease activity of different complexes to their DNA polymerase activity varies from 0.5 to 12. Other cases of discovery of the complexes of DNA polymerases with 3'----5' exonucleases are discussed. It is suggested that the absence of 3'----5' exonuclease active site in the DNA polymerase polypeptide is compensated by the complex formation of the corresponding enzymes.